Ceacam1 mediated protective immunity

ABSTRACT

The presently described technology relates to the modulation of specific immune responses to create a protective immunity in the treatment of autoimmune diseases and diseases requiring the transplantation of tissue. In particular, the present technology relates to the suppression of immune responses in a targeted fashion, by increasing the functional concentration of the CEACAM1 protein in a target tissue to create a localized protective immunity for the treatment of autoimmune diseases and diseases requiring the transplantation of tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCE

The present application is a continuation of U.S. patent applicationSer. No. 14/287,901, which was filed May 27, 2014. U.S. patentapplication Ser. No. 14/287,901 relates to and claims priority benefitsfrom U.S. patent application Ser. No. 11/423,395, filed Jun. 9, 2006.U.S. patent application Ser. No. 11/423,395 relates to and claimspriority benefits from U.S. Provisional Application Ser. No. 60/689,316,with attorney docket number 16667US01, filed Jun. 9, 2005, and titled“MODULATION OF IMMUNITY AND CEACAM1 ACTIVITY,” the contents of which arehereby incorporated herein by reference in their entirety. Additionally,all cited references in the present application are incorporated byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to the modulation of the immune system in general.More specifically, certain aspects of the present invention relate tothe modulation of specific immune responses to create a protectiveimmunity in the treatment of autoimmune diseases and diseases requiringthe transplantation of tissue.

BACKGROUND OF THE INVENTION

The human carcinoembryonic Ag (CEA)3 protein family encompasses severalforms of proteins with different biochemical features. These proteinsare encoded by 29 genes tandemly arranged on chromosome 19q13.2. CEAfamily genes have been classified into two major subfamilies, the CEAcell adhesion molecule (CEACAM) and the pregnancy-specific glycoproteinsubgroups. The CEACAM proteins, which are part of the larger Igsuperfamily, include CEACAM1, -3, -4, -5, -6, -7, and -8. They share acommon basic structure of sequentially ordered different Ig-likedomain(s) and are able to interact with each other. For example, it hasbeen reported that various CEACAM proteins, such as CEACAM1 or CEACAM5,exhibit both homophilic and heterophilic interactions.

CEACAM1 (CD66a), a transmembrane protein and member of thecarcinoembryonic Ags family, contains two ITIM sequences located withinits cytosolic tail. CEACAM1 interacts with other known CD66 proteins,including CD66a, CD66c, and CD66e proteins. It is expressed on a widespectrum of cells, ranging from epithelial to hemopoietic origin. AmongCD66 proteins tested, only the CD66a protein is expressed on the surfaceof activated CD16-negative NK cells.

The various CEACAM proteins have different biochemical features,including but not limited to anchorage to cell surface (GPI-linked,transmembrane or secreted forms), length of cytoplasmic tail (long orshort), and the presence or absence of various signal transductionmotifs. These proteins are actively involved in numerous physiologicaland pathological processes.

CEACAM1 is a transmembrane protein that can be detected on some immunecells as well as on epithelial cells. Many different functions have beenattributed to the CEACAM1 protein. It was shown that the CEACAM1 proteinexhibits antiproliferative properties in carcinomas of colon, prostate,as well as other types of cancer. Additional data support the centralinvolvement of CEACAM1 in angiogenesis and metastasis. CEACAM1 also hasa role in the modulation of innate and adaptive immune responses. Thepresent inventor has shown that CEACAM1 homophilic interactions inhibitNK-mediated killing activity independently of MHC class I recognition.This novel mechanism plays a pivotal role in the inhibition of activateddecidual lymphocytes in vitro and most likely also in vivo afterinfection, including for example CMV infections. The CEACAM1 homophilicinteractions are possibly important in some cases of metastaticmelanoma, as increased CEACAM1 expression was observed on NK cellsderived from some patients compared with healthy donors. There is anassociation of CEACAM1 expression on primary cutaneous melanoma lesionswith the development of metastatic disease and poor survival. Thepresent inventor has demonstrated the role of CEACAM1-mediatedinhibition in maintaining NK self-tolerance in TAP2-deficient patients.Additional reports have indicated that CEACAM1 engagement either by TCRcross-linking with mAb or by Neisseria gonorrhoeae Opa proteins inhibitsT cell activation and proliferation.

The CEACAM1 protein interacts with other CEACAM protein family members,such as CEACAM1 itself and CEACAM5. At least part or the entire bindingsite of human CEACAM1 is located at the N-terminal Ig-V-type domain ofthe CEACAM1 protein. In particular, amino acids 39V and 40D and the saltbridge between 64R and 82D may play an important role in this binding.Most amino acid sequences of the N-terminal domain of CEACAM1, -3, -5,and -6 are identical, and predicted binding residues are conserved amongthe four proteins. These proteins might interact with each other. Thisis of particular importance, because in certain tumors the CEACAM1protein is down-regulated, followed by upregulation of CEACAM6 proteinexpression.

The present inventor has demonstrated the inability of CEACAM1 to bindCEACAM6. The present inventor has also directly shown that the presenceof both residues 43R and 44Q in the CEACAM1 is crucial for thehomophilic CEACAM1 interaction and that substitution of these residueswith the 43S and 44L residues that are present in CEACAM6 abolishes theinhibitory effect. The reciprocal substitution of 43S and 44L of CEACAM6to the 43R and 44Q residues, respectively, results in the gain ofinhibitory heterophilic interactions with the CEACAM1 protein. Thedichotomy of CEACAM family members by recognition of CEACAM1 isdetermined by the presence of R and Q at positions 43 and 44.

Natural killer (NK) cells belong to the innate immune system andefficiently kill virus-infected and tumor cells. NK killing is generallyrestricted mainly to cells that have lost class I MHC expression, aphenomenon known as the missing self. NK cell cytotoxicity is tightlyregulated by various inhibitory class I MHC-recognizing receptors. Theinhibitory signal is delivered via the immuno-receptor tyrosine-basedinhibitory motif (ITIM) sequences found within the cytosolic tail ofthese receptors. Families of class I MHC binding inhibitory receptorsinclude members of the Ig superfamily, namely killer Ig-relatedtwo-domain long-tail (p58) and three-domain long-tail (p70) receptors,the C-type lectin complex CD94/NKG2A, and the leukocyte Ig-like receptor(Ig-like transcript) family.

There are also other NK-specific receptors, termed natural cytotoxicityreceptors (NCRs), which are directly involved in triggering NK cellcytotoxicity. The NCR group consists of several proteins, includingNKp30, NKp44, NKp46, NKp80, and CD16. The cellular lysis ligands for allthe NCRs have yet to be identified. A viral ligand (hemagglutinin) wasshown to interact with the NKp46 receptor, and this interaction resultedin the enhancement of lysis of certain virus-infected cells. Indeed, thekilling activity of target cells by human natural killer (NK) cells ismediated via a panel of lysis receptors of which is included CD16,NKp30, NKp44, NKp46, and NKG2D. These receptors recognize viral ligandssuch as hemagglutinin, stress-induced ligands such as MHC class Ichain-related antigen A (MICA) and MICB, or other as-yet-undefined,cellular ligands. As mentioned, cells are protected from lysis by NKcells mainly owing to the interactions between class I MHC proteins andthe appropriate inhibitory NK receptors.

The present inventor has identified a novel class I MHC-independentinhibitory mechanism of human NK cytotoxicity, mediated via thecarcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)homophilic interactions. Furthermore, the present inventor has foundthat the CEACAM1 protein plays a pivotal role in the inhibition ofkilling, proliferation, and cytokine secretion of interleukin 2(IL-2)-activated decidual NK, T, and NKT cells, respectively.

Once class I MHC proteins are removed from the cell surface, these cellsbecome susceptible to NK cell attack. It was surprising to learn thatpatients with transporter associated with antigen processing (TAP2)deficiency do not frequently suffer from autoimmune manifestations atearly stages of their life. Activated NK cells derived from suchpatients may either be expressing an unknown inhibitory mechanism or aremissing an unidentified lysis receptor. NK tolerance toward self-cellsmight be controlled by similar mechanisms.

The present inventor has demonstrated that the expression of the NKp46receptor is severely impaired in a newly identified TAP2-deficientfamily and that the vast majority of activated NK cells derived fromthese patients use the CEACAM1 protein interactions to avoid tumor andautologous cell killing.

The present inventor has also found that CD16-negative NK clonesinefficiently kill 1106mel cells because of the CD66a homotypicinteractions The inhibition of NK cell cytotoxicity by CD66a wasdependent on the level of CD66a expression on both effector and targetcells. 721.221 cells expressing CD66a protein were protected from lysisby CD66a-expressing NK and YTS cells. Redirected lysis experimentsperformed by the present inventors showed that the strength of theinhibition is dependent on the level of CD66a expression on NK cells. Adramatic increase in CD66a expression was observed among NK cellsisolated from melanoma patients. As stated above, a novel class IMHC-independent inhibitory mechanism of human NK cell cytotoxicity hasbeen demonstrated by the present inventors. Some melanoma tumors may usethis mechanism to avoid attack by NK cells.

Human natural killer (NK) cells are able to eliminate a broad spectrumof tumors and virus-infected cells by using several receptors, such asCD16, NKp30, NKp44, NKp46 and NKG2D. These receptors recognize eitherviral ligands, such as hemagglutinin, stress induced ligands, such asMICA and MICB, or other yet-undefined cellular ligands. Other NKreceptors mediate inhibition of the killing activity followinginteraction with MHC class I proteins present on normal cells. Removalof MHC class I proteins from the cell surface renders it susceptible toNK cell attack through the phenomenon known as the “missing self”.

Additional receptors are also able to manipulate NK cell cytotoxicityand the present inventors have shown a novel MHC class I independentinhibitory mechanism of human NK cytotoxicity that is mediated by theCEACAM1 homophilic interactions. This CEACAM1-mediated inhibition mightplay an important role in the in vivo development of melanoma in humanpatients. A 10-year follow-up study correlated the presence of CEACAM1on primary melanoma lesions with poor survival. In addition, the presentinventors have demonstrated the pivotal role of the CEACAM1 in theinhibition of killing, cytokine secretion and proliferation of activateddecidual NK, NKT and T cells, respectively. The present inventors havealso provided substantial evidence for a major role of the inhibitoryCEACAM1 interactions in controlling NK cell autoreactivity inTAP2-deficient patients.

The presence of human soluble CEACAM1 protein can be observed in theserum of healthy donors. Furthermore, variations in serum levels of thesoluble CEACAM1 protein are observed in various pathologies. Forexample, increased CEACAM1 levels were observed in the sera of patientswith various hepatic diseases such as obstructive jaundice, primarybilliary cirrhosis, autoimmune hepatitis and cholangiocarcinoma. Adecrease in the soluble CEACAM1 level has not been reported.

The present inventor has shown that the soluble CEACAM1 protein blocksthe CEACAM1-mediated inhibition of NK cell killing activity in adose-dependent manner. Moreover, the present inventors have demonstratedthat serum CEACAM1 levels among the TAP2-deficient patients aredecreased when compared to normal individuals. These findings concurwith the dominant role of the CEACAM1-mediated inhibition in controllingNK autoreactivity in TAP2-deficient patients. Thus, the maximalcompensatory effect of CEACAM1-mediated inhibition is attained.

At least one object of the present invention is the modulation ofCEACAM1 activity to effect control over the immune system and inparticular specific immune responses in the treatment of disease. Inparticular, the present invention relates to the supression of specificimmune responses, by increasing the functional concentration of theCEACAM1 protein, to create a protective immunity in the treatment ofcertain disease states, including but not limited to autoimmune diseasesand diseases requiring the transplantation of tissue.

Autoimmune disease results when the immune system mistakes self tissuesfor nonself and mounts an inappropriate attack. There are many differentautoimmune diseases. Autoimmune disease can affect many parts of thebody, including but not limited to nerves, muscles, the endocrine system(system that directs your body's hormones and other chemicals), and thedigestive system. Some examples are Wegener's granulomatosis, multiplesclerosis, type 1 diabetes mellitus, rheumatoid arthritis, and Crohn'sdisease.

Transplant rejection occurs when the immune system of the recipient of atransplant attacks the transplanted organ or tissue. This is because anormal healthy human immune system can distinguish foreign tissues andattempts to destroy the transplant, just as it attempts to destroyinfective organisms such as bacteria and viruses.

At present, regimens to treat both autoimmune diseases and tissuetransplant rejection employ general immunosuppressant drugs. The presentinvention relates to the supression of immune responses in a specificfashion, by increasing the functional concentration of the CEACAM1protein in a target tissue to create a localized protective immunity forthe treatment of autoimmune diseases and diseases requiring thetransplantation of tissue.

BRIEF SUMMARY OF THE INVENTION

One object of the present invention is to provide methods andcompositions for the modulation of the immune system and/or one or morespecific immune responses. Another object of the present invention is toprovide methods and compositions for the regulation of lymphocyteactivity. A further object of the present invention is to providemethods and/or compositions for inducing a tolerogenic state(immunologic tolerance or protective immunity) in a specified tissue,including but not limited to tissue affected by autoimmune disease ortissue being prepared for transplantation. A still further object of thepresent invention is to provide methods and compositions for theregulation of the immune system and specific immune responses in thetreatment of disease, including but not limited to autoimmune diseasesand diseases requiring organ transplantation.

One or more of the preceding objects, or one or more other objects whichwill become plain upon consideration of the present specification, aresatisfied by the invention described herein.

One aspect of the present invention that satisfies one or more of thepreceding objects provides methods for inducing a protective immunity ina target tissue. A further aspect of the present invention providesmethods for the induction of CEACAM1 protein production in the tissuetargeted for induction of a protective immunity. One embodiment of thisaspect of the present invention comprises methods for the induction ofCEACAM1 protein production in the tissue targeted for induction of aprotective immunity.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. CEACAM1-Ig does not recognize the CEACAM6 protein. Stable.221/CEACAM1 and .221/CEACAM6 were generated as described. Theexpression level was monitored with the Kat4c mAb (empty histograms).Binding of CEACAM1 was assessed with the CEACAM1-Ig fusion protein(empty histograms). The reagents used are indicated in each histogram.The background (shaded histograms) is the corresponding staining of .221parental cells. This figure shows one representative experiment of 20performed.

FIG. 2. CEACAM1 and the CEACAM6 proteins do not functionally interact.A, The amount of mIL-2 in culture supernatant of Kat4ctreated andcontrol 12E7 BW/CEACAM1-_ cells as measured by ELISA. The x-axis is theamount of immobilized mAb per reaction, and the y-axis is the opticdensity at a wavelength of 650 nm. This figure shows the mean of threeindependent experiments. B, mIL-2 secretion by BW parental cells or byBW/CEACAM1-_ cells co incubated for 48 h with irradiated .221,0.221/CEACAM1, or with .221/CEACAM6 cells. The y-axis is the opticdensity at a wavelength of 650 nm. The average of four independentexperiments is shown.

FIG. 3. Substitution of the GPI link of CEACAM6 with the transmembraneand tail of CEACAM1 does not induce heterophilic binding. A, Staining of.221/CCM6-TailCCM1 cells with Kat4c (empty histogram) mAb or CEACAM1-Ig(empty histogram). The reagents used are indicated in each histogram.The background is the staining of .221 parental cells (shadedhistogram). This figure shows one representative experiment of 10performed. B, mIL-2 secretion by BW/CEACAM1-cells co incubated for 48 hwith irradiated .221 or with .221 transfectants. The y-axis indicatesthe optic density at a wavelength of 650 nm. The average of fourindependent experiments is shown.

FIG. 4. Sequence alignment of CEACAM family members. Letters in boldindicate amino acid residue 1. Identical residues of the known motifscrucial for binding are underlined. Different residues in the bindingmotifs are highlighted with black (for RQ residues) or gray (for SLresidues) backgrounds.

FIG. 5. Recognition of CEACAM1 is dependent on the presence of 43R44Q.A, Staining of .221/CCM1-RQ43,44SL or .221/CCM6-SL43,44RQ cells withKat4c mAb or CEACAM1-Ig fusion protein as indicated in each histogram.The corresponding staining of .221 parental cells was used as background(shaded histograms). This figure shows one representative experiment ofsix performed. B, Staining of .221/CCM1-RQ43,44SL or .221/CCM6-SL43,44RQcells with the conformation-dependent 5F4 mAb (thick lines). Thecorresponding staining of .221 parental cells was used as background(thin lines). This figure shows one representative experiment of sixperformed. C, mIL-2 secretion by BW/CEACAM1-_ cells co incubated for 48h with irradiated .221 or .221 transfectants. The y-axis indicates theoptic density at a wavelength of 650 nm. The average of five independentexperiments is shown.

FIG. 6. NK-mediated cytotoxicity. CEACAM1-positive NK clones wereobtained as described in Materials and Methods. NK clones were tested inkilling assays against the indicated cells in an E:T cell ratio of 2:1.When rabbit polyclonal Abs were included in the assays, the finalconcentration was 20 _ g/ml. This figure shows the results of arepresentative NK clone.

FIG. 7. Both the 43 and 44 residues of CEACAM1 are crucial for theinteraction. A, Staining of .221 and various .221 stable transfectantswith 5F4 mAb (u) or with Kat4c (f). The staining of the secondaryreagent FITC-conjugated goat anti-mouse F(ab_)2 of each cell type wasused as background (_). The y-axis indicates the median fluorescenceintensity (MFI). This figure shows one representative experiment of fourperformed. B, Staining of .221 and various .221 stable transfectantswith CEACAM1-Ig fusion protein. The y-axis indicates the medianfluorescence intensity (MFI). This figure shows one representativeexperiment of four performed. C, YTS cells expressing the CEACAM1protein (YTS/CCM1) or mock-transfected (YTS/control) were tested inkilling assays against .221 and .221 transfectants. The E:T cell ratiowas 2:1. This figure shows the average of three independent experiments.D, Staining of .221/CEACAM5 cells with 5F4, Kat4c, or CEACAM1-Ig wasperformed as indicated in each histogram. The corresponding staining of.221 parental cells was used as background (thin lines). This figureshows one representative experiment of six performed.

FIG. 8. Family pedigree of the TAP2-deficient patients. Patients areindicated as black symbols, the parents as gray symbols and all otherhealthy family members as white symbols. Cousin marriage is representedby a double line. Roman numerals indicate the generations, whereasArabic numerals indicate individuals.

FIG. 9. PBL characterization of TAP2-deficient patients. (A) PBLobtained from the patients and from the healthy sister was stained forCD3 and CD56. (B) Staining of PBL obtained from the patients and fromthe healthy sister for CD3, CD56 and CD16. The dot plots analysispresented shows CD16 and CD56 expression on already gated NK cells. Thevertical dashed lines discriminate CD56dim from CD56bright. For (A) and(B) one representative experiment out of three performed is shown.

FIG. 10. Lack of recognition of the patients' cells by KIR2DL2-Ig andLIR1-Ig. The various EBV cell lines were stained with mAbW6/32 or withvarious Ig-fusion proteins. The secondary F(ab′)2 detection antibodiesalone were used as background. One representative experiment out of fourperformed is shown.

FIG. 11. High expression of CEACAM1 on activated TAP2-deficient NKcells. NK clones expressing CEACAM1 were divided into groups accordingto expression level of CEACAM1 (indicated on the left). CEACAM1expression on one representative NK clone of each group is shown. Thepercentages of the NK clones similar to the NK clone presented in eachdonor are indicated in each histogram.

FIG. 12. CEACAM1-mediated inhibition of NK killing activity is blockedby the soluble CEACAM1-Ig. (A) Bulk NK cultures were stained for CD16,CD56 and CEACAM1. Contour plot X-axis is CEACAM1 log fluorescence andY-axis is CD16 or CD56 log fluorescence. One representative experimentout of two performed is shown. (B-D) Killing of .221 and .221/CEACAM1cells, incubated with various amounts of the CEACAM1-Ig (CCM1-Ig) fusionprotein or the anti-CEACAM antibodies. Killing assays were performedwith NK-B cells (B), CEACAM1-NK-M cells (C) or CEACAM1+NK-Y cells (D).The E:T ratio was 2:1. For (B-D), the average of three independentexperiments is shown.

FIG. 13. Decreased level of soluble CEACAM1 protein in the serum ofTAP2-deficient patients. (A) Serum samples were analyzed for thepresence of soluble CEACAM1 by ELISA. X-axis indicates the amount ofdetected soluble CEACAM1. The mean of three independent experiments isshown. (B-D) Killing of .221 and .221/CEACAM1 pre-incubated either withno serum, with serum derived from patient B (Serum B) or from a healthydonor (Serum Healthy). Killing assays were performed with NK-B (B),NKHealthy (C) and with NK-Sister (D). The E:T was 2:1.

FIG. 14. Cell surface CEACAM1 level is not regulated by MBMP activity.Surface expression of the MHC class I (A, B) on NK cells derived fromthe mother (NK M), healthy donor (NK Y) or patient B (NK B), NKp46 (C)and the CEACAM1 protein (D). Cells were analyzed by FACS using the W6/32(A, B), 461-G1 (C) and the Kat4c mAb (D). The tested protein isindicated on the Y-axis of each plot. Expression was analyzed followingstimulation with PMA and Ca2+ ionophore. These experiments wereperformed either with or without the MBMP inhibitor BB-94 (indicated inthe bottom of each plot). Average results of three independentexperiments are shown.

FIG. 15. The reduction in class I MHC expression is due to TAP2deficiency. Fusion of EBV-A, EBV-B, and EBV-C with various B-cell linesdefective either for the TAP1 and TAP2 subunits (0.174) or none of them(0.45). Total mixture of cells was analyzed by FACS. Fused cells wereidentified by HLA-A3 expression. Staining with HLA-A3 is on the y-axis,and forward scatter is on the x-axis. One representative experiment isshown of 3 performed.

FIG. 16. Impaired expression and function of NKp46 on freshly isolatedNK cells. (A) NKp46 expression on freshly isolated bulk NK cells.Staining was detected by mAb 461-G1 in the form of F(ab′)₂, and the MFIstaining is indicated in each histogram. One representative experimentis shown of 3 performed. (B) Killing of .221 cells by freshly isolatedNK cells derived from indicated donors. The mean results of 3independent experiments are shown. The data represent means of thepercentage of killing±SDs.

FIG. 17. Inhibition of NK-mediated killing by homophilic CEACAM1interactions. Killing of .221 and .221/CEACAM1 cells, incubated with orwithout polyclonal anti-CEACAM antibodies, by a representative CEACAM1⁺NK clone (panel A) or by a CEACAM1⁻ NK clone (panel B). As control,anti-glutathion S-transferase (GST)-ABL polyclonal antibodies were used.The effector-to-target (E/T) ratio was 2:1. All antibodies used were inthe form of F(ab′)₂. Figures show the average of 3 independentexperiments. The data represent means of the percentage of killing±SDs.

FIG. 18. Killing of PHA-induced T-cell blasts. (A) Staining ofPHA-induced T-cell blasts with various mAbs. Staining of PHA-inducedT-cell blasts derived from patient A and from the healthy sister wasperformed with the F(ab′)₂ fragments of anti-CD3, anti-CEACAM1, andanti-MHC class I mAb HP-1F7. (B) Staining of PHA-induced T-cell blastsand of the LnCap cell line with various fusion proteins. Staining wasperformed with the NKp46-Ig, NKp30-Ig, NKp44-Ig, and the control CD99-Igfusion proteins. (C) NK clones derived from patients A, B, and C wereassayed for cytotoxic activity against autologous PHA-induced T-cellblasts. The NK clones obtained from the healthy sister were assayedagainst PHA-induced T-cell blasts derived from patient A. NK clones werepreincubated with or without F(ab′)₂ fragments of polyclonal anti-CEACAMor the control polyclonal antiubiquitin antibodies. The targets,autologous PHA-induced T-cell blasts, were incubated with or without theF(ab′)₂ fragments of HP-1F7 or the control 12E7 mAb. Assays wereperformed at an E/T ratio of 2:1. Shown are the mean results of severalNK clones that were obtained from 3 independent experiments. The datarepresent the mean percentage of killing±SD. (D) NK clones derivedeither from the healthy sister or from patients A, B, and C were assayedfor killing of PHA-induced T-cell blasts derived from the healthysister. NK clones and target PHA-induced T-cell blasts were pretreatedas described for panel C. Assays were performed at an E/T ratio of 2:1.Shown are the mean results of several NK clones that were obtained from3 independent experiments. All mAbs used were in the form of F(ab′)₂.The data represent the mean percentage of killing±SD.

FIG. 19. Killing of melanoma lines by NK clones. Lysis of 1106mel cells(A-D) and 1259mel cells (E) by CD16⁺CD66a⁻ NK clone (A and B) orCD16⁻CD66a⁺ NK clone (C-E) was performed as described in Materials andMethods of this section. The anti-CD99 mAb (12E7) and anti-CD66apolyclonal Abs were incubated with the target cells (A and C) or withthe effector cells (B, D, and E). The E:T cell ratio was 3:1.

FIG. 20. Expression of CD66a on various cell types. Transfectants weregenerated as described in Materials and Methods of this section. Shownis CD66a staining of transfected .221 and YTS cells with the anti-CD66amAb Kat4c (dark line) overlaid on the staining of the parental cells(.221 and YTS) with the same mAb (light line). Staining of arepresentative NK clone by Kat4c (dark line) overlaid on the staining ofthe same NK clones with the control FITC-conjugated goat anti-mouse Abs(light line) is also shown. The figure shows one representativeexperiment of three performed.

FIG. 21. Killing of various 721.221 transfectants by various YTStransfectants. Killing assays were performed as described in thissection. The various YTS transfectants are indicated in each histogram.The figure shows one representative experiment of six performed.

FIG. 22. Killing of .221/CD66a^(high) cells by NK clones. Killing oftarget cells by YTS/CD66a (A), CD66-positive NK clone (B), andCD66-negative NK clone (C) incubated with or without anti-CD66apolyclonal Abs. The figure shows one representative experiment of fiveperformed.

FIG. 23. The high level of CD66a expression on NK clones correlates withefficient inhibition of redirected lysis of P815 cells. The CD66aexpression on NK clones was monitored by FACS. To correctly compare thelevel of CD66a expression among different NK clones, and because thebackground staining F(ab)′₂ of FITC-conjugated goat anti-mouse IgG Absof each NK clone might be different, the level of CD66a expression ineach clone was determined by dividing the MFI of the CD66a staining on agiven clone with the MFI of the background staining of the same clone.The fold increase in CD66a staining above the background of each cloneis indicated in brackets. The percent inhibition of each clone wascalculated by dividing the percentage of specific lysis of the NK cloneincubated with anti-CD66 mAb by that of the clone incubated with no mAb.Similar results were obtained when the specific lysis of each NK cloneincubated with anti-CD66 mAb was divided by the percent specific lysisof the same NK clone incubated with control mAb. The NK clones arepresented in the figure in the order of the fold increase in CD66a abovebackground. The figure shows CD16⁻CD66⁻ clones (24, 89, and 98),CD16⁻CD66⁺ clones (21, 79, 84, and 100), CD16⁺CD66⁻ clones (25, 47, 48,63, and 64), and CD16⁺CD66⁺ clones (1, 2, 3, 9, 10, 13, 17, 30, 32, 34,43, 44, 49, 58, 61, 65, 69, 70, 71, 73, 75, and 96). When CD16⁻ NKclones were used, anti-NKp44 and NKp46 sera were included to stimulatethe redirected lysis experiments. When CD16⁺ NK clones were used,anti-CD16 mAb was included in the redirected lysis experiments. Thefigure shows NK clones generated from one healthy donor YF that containsan unusually high number of CD16⁺CD66⁺ NK clones.

FIG. 24. CD66a expression on NK cells derived from healthy donors andmelanoma patients. Lymphocytes were obtained from surgically removedlymph nodes derived from two different melanoma patients, infiltratedwith melanoma metastases positive (A) or negative (C) for CD66aexpression. Lymphocytes were also obtained from peripheral blood ofanother melanoma patient (B) or from peripheral blood of representativehealthy donor (D). Lymphocytes were stained for expression of CD3, CD16,CD56, and CD66 as described in Materials and Methods of this section.The figure shows CD66a expression on NK cells.

FIG. 25. CEACAM1 staining of decidual lymphocytes. Decidual lymphocyteswere isolated and quadruple-stained as described in Methods. (a-c)CEACAM1 staining on nonactivated decidual NK cells (a), T cells (b), andNKT cells (c). One representative experiment is shown out of threeperformed. Decidual lymphocytes were cultured in the presence of IL-2 asdescribed (20) and then screened for CEACAM1 expression with the 5F4mAb. (d-f) CEACAM1 staining for activated decidual NK clone (d), T clone(e), and NKT clone (f). Similar results were obtained when otherlymphocyte clones were used. (g and h) Staining of EVTs for HLA-G andCEACAM1, respectively. Bold lines represent mAb staining and thin linesshow background staining.

FIG. 26. Staining of .221 cells expressing various members of the CEACAMfamily using specific anti-CEACAM antibodies. .221 transfectants weregenerated as described in Methods. Each row shows the staining performedon a particular transfectant (indicated at left), and each column showsthe staining with a particular antibody (indicated at top). Bold linesrepresent antibody staining and thin lines show background staining on.221 cells. One representative experiment is shown out of threeperformed.

FIG. 27. CEACAM1-mediated inhibition of decidual NK cytotoxicity.Decidual NK clones were stained for CEACAM1 expression. (a) CEACAM1staining of decidual NK clone 17 using the anti-CEACAM1 mAb 5F4 (boldline). The thin line shows the control staining. (b) Killing andinhibition of NK clone 17 by .221 cells and by .221 cells transfectedwith CEACAM1 (.221/CEACAM1). Blocking experiments were performed using40 □l/ml of anti-CEACAM antibodies. Average of three independentexperiments is shown. Similar results were obtained when otherCEACAM1+NK clones were used.

FIG. 28. CEACAM1-mediated interactions inhibit SEB-induced T cellproliferation. Decidual T cell clones were tested for expression of CD4(a), V□17 (b), and CEACAM1 (c) by flow cytometry. Bold lines indicatemAb staining and thin lines indicate control staining. (d) Fiftythousand cells of the presented T cell clone were incubated for 2 dayswith 25,000 irradiated .221 cells or with .221 cells transfected withCEACAM1 (.221/CEACAM1), in the presence of decreasing SEB concentrationsas indicated in the figure. Proliferation was measured with 3H-thymidineincorporation. The figure represents the average of ten independentexperiments. Similar results were obtained when other T cell clones wereused.

FIG. 29. CEACAM1-mediated inhibition of IFN-□□ secretion from NKT cells.(a) CEACAM1 expression on isolated activated NKT clone. The bold lineshows the staining with 5F4 mAb, and the thin line shows the controlstaining. (b) The amount of IFN-□□ in culture supernatant of mAb-treatedand untreated NKT clone cells measured by ELISA. The average of twoindependent experiments is shown. Cross-linking of surface CEACAM1 wasperformed without (c) or with (d) the Kat4c mAb, and intracellularstaining for IFN-□□ was performed. One representative experiment isshown out of two performed. Similar results were obtained when other NKTcell clones were used.

FIG. 30. CEACAM1-Ig specifically binds to CMV-infected fibroblasts. (a)Binding of CEACAM1-Ig to .221/CEACAM1 cells (bold line) but not toparental .221 (thin line). The figure shows a representative experimentout of three performed. (b) Day-by-day staining of uninfected andCMV-infected HFF cells in the presence or absence of 300 □g/ml of theantiviral agent PFA. Cells were stained with CEACAM1-Ig and with thecontrol CD99-Ig fusion protein as described in Methods. Data arepresented as fold increase above the staining of uninfected cells. Theaverage of two independent experiments is shown.

FIG. 31. The functional interactions between BW/CEACAM1□□ andCMV-infected HFFs elicit IL-2 secretion. (a) Spontaneous IL-2 secretionby BW and various BW transfectants after 48 hours of incubation. Theaverage of 20 independent experiments is shown. (b) IL-2 secretion byBW/CEACAM1□□ cells coincubated for 24 hours with irradiated .221 or with.221/CEACAM1 cells. The average of six independent experiments is shown.(c) IL-2 secretion after coincubation of BW or BW/CEACAM1□□ cells withuninfected or CMVinfected HFF cells for 48 hours. No IL-2 secretionabove background levels was observed when PFA was included in the assay(only day 6 is shown). Experiments were performed concomitantly with theflow cytometry binding assays of CEACAM1-Ig shown in FIG. 29. Theaverage of two independent experiments is shown.

FIG. 32. CMV isolated from infected decidua induces a ligand for theCEACAM1 on infected HFF cells. (a) Staining of HFF cells infected withclinical CMV strain with CD99-Ig or with CEACAM1-Ig. No staining wasobserved when proteins were omitted, indicated by the horizontal line.FSC, forward scatter. (b) IL-2 secretion from BW or BW/CEACAM1□□ cellscoincubated with HFF-infected cells for 48 hours. The average of twoexperiments is shown.

DETAILED DESCRIPTION OF THE INVENTION

While the present invention has been described with reference to certainembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted withoutdeparting from the scope of the present invention. In addition, manymodifications may be made to adapt a particular situation or material tothe teachings of the present invention without departing from its scope.Therefore, it is intended that the present invention not be limited tothe particular embodiment disclosed, but that the present invention willinclude all embodiments falling within the scope of the appended claims.

One aspect of the present invention provides methods for inducing aprotective immunity in a target tissue. One embodiment of this aspect ofthe present invention comprises methods for the induction of CEACAM1protein production in the tissue targeted for induction of a protectiveimmunity. The target tissue includes but is not limited to tissueafflicted by an autoimmune disease and/or tissue being prepared fortransplantation. In preferred embodiments of this aspect of the presentinvention the induction of CEACAM1 protein production comprises theactivation of CEACAM1 gene expression. activation of CEACAM1 geneexpression can be accomplished any number of techniques known to thoseskilled in the art for the induction of gene expression, including butnot limited to those techniques comprising contacting the target tissuewith a signal transduction protein, transcriptional activator protein,nucleic acid, small molecule compound, or combination thereof. Inpreferred embodiments of this aspect of the present invention, theinduction of CEACAM1 protein production comprises the transfer of anucleic acid sequence into the cells of the target tissue encoding theCEACAM1 protein, or another protein that directly or indirectlyincreases CEACAM1 gene expression. The transfer of nucleic acid into aselected target tissue pursuant to this embodiment of the presentinvention can be accomplished by any number of techniques known to thoseskilled in the art including but not limited to viral-mediated transfer,particle-mediated transfer, or magnetic cationic liposome mediatedtransfer.

The presently described technology provides methods and compositions forthe regulation of the immune system and specific immune responses, andin particular to methods and compositions for the regulation oflymphocyte activity. One aspect of the present invention is thefunctional modulation of at least one member of the CEACAM proteinfamily, said CEACAM protein being either membrane bound or free. TheCEACAM protein family, which are part of the larger Ig superfamily,include without limitation CEACAM 1, -3, -4, -5, -6, -7, and -8. TheCEACAM protein family share a common basic structure of sequentiallyordered different Ig-like domain(s) and are able to interact with eachother.

In certain embodiments of the presently described invention, regulationof the immune system and/or one or more specific immune responsescomprises the positive modulation of CEACAM1 gene expression ortranslation of CEACAM1 mRNA. The positive modulation of CEACAM1 geneexpression or CEACAM1 mRNA translation can comprise any number oftechniques know to those skilled in the art for the modulation of geneexpression, and can involve contacting any cell or grouping of cells(e.g. tissue) with a protein, peptide, peptidomimetic, nucleic acid,nucleic acid analog, small molecule, or some combination thereof.

In a further aspect of the presently described invention, there areprovided methods and/or compositions for modulating the immune systemand/or one or more specific immune responses in the course of treating adisease. Exemplar diseases include but are not limited to autoimmuneconditions, and those diseases requiring tissue transplantation.

Certain aspects of the present invention can be performed in anyenvironment including but not limited to in situ, in vivo, or in vitroenvironments. For example, the methods and/or compositions of thepresent invention can be employed in a cell culture or in the livingbody of an animal, such as a human.

In a still further aspect of the presently described invention, methodsand/or materials are provided for inducing a tolerogenic state(immunologic tolerance) in a specified tissue. As used herein, oneexemplar definition for tissue includes any aggregate of cells. Thespecified tissue may include tissue affected by an autoimmune disease ortissue being prepared for transplantation. In one preferred embodimentthereof, the induction of the tolerogenic state includes the stimulationof CEACAM1 gene expression and protein production. This can beaccomplished by any number of techniques know to those skilled in theart for the enhancement of gene expression and protein production.

At least one of the objects of the present invention is to induce atolerogenic state in a specified tissue. This aspect of the presentinvention for example includes the induction of CEACAM1 proteinproduction. The specified tissue may be tissue affected by an autoimmunedisease or tissue being prepared for transplantation. The induction ofCEACAM1 protein production includes, for example, the generation ofCEACAM1 gene expression. In at least on aspect of the present invention,induction of CEACAM1 protein production includes the transfer of geneticmaterial into the cells of the specified tissue for which a protectiveimmunity is to be generated. The genetic material, for example, can becomposed of a CEACAM1 family gene. Cis acting genetic elements may alsobe added to facilitate, for example, the integration of the geneticmaterial into the genome of the specified cells, or the production ofCEACAM1 protein, or both. The cis acting genetic elements may includegenetic material effective in inducing efficient gene expression,efficient translation, increased recombination frequency, increasedtargeted recombination, or some combination thereof.

In an additional aspect of the invention, materials and/or methods areprovided for inducing a protective immunity or tolorogenic state in aspecified tissue, which includes the induction of CEACAM1 proteinproduction by transferring genetic material that includes a gene whoseprotein product induces the increased production of a CEACAM1 familyprotein. For example, the gene whose protein product induces theincreased production of a CEACAM1 family protein may be a transcriptionfactor, including for example a transcriptional activator.

One aspect of the present invention provides methods and/or materialsfor imparting a tolerogenic state (i.e.—immunologic tolerance orprotective immunity) upon a specified tissue. One embodiment of thisaspect of the present invention provides methods and/or materials forimparting a tolerogenic state upon a specified tissue by imparting uponor inducing within a specified tissue CEACAM1 function. The specifiedtissue may be any tissue upon which it is desirable to create atolerogenic state. For example, the tissue may be tissue that is beingprepared for transplantation, or the tissue may be tissue which isafflicted by autoimmune disease. One definition of a tolerogenic stateis a state characterized by an immunologic tolerance.

A further aspect of the present invention provides methods and/ormaterials for preparing tissue for grafting or transplantation. In oneembodiment, the present invention provides materials and/or methods formitigating the potential for immunological rejection of grafted ortransplanted tissue. For example, the present invention provides forincreased transplant tolerance strategies that would thwart theimmunological rejection of transplanted or grafted tissue by impartingupon the transplanted tissue a tolerogenic state (immunologictolerance), while preserving a body's general immune competence,including for example normal immune responses to pathogens and cancerrisks. This aspect of the present invention may be accomplished, atleast in part, by conveying or imparting CEACAM1 function or activityupon tissue to be transplanted or grafted. For example, tissue to betransplanted or grafted can be transformed or transfection of withgenetic material effective in facilitating or inducing the production ofCEACAM1 protein. This can be performed, for example, by the transfer ofgenetic material that is effective in inducing CEACAM1 proteinproduction to tissue being prepared for transplantation or grafting. Thegenetic material that is transferred may include, for example, one ormore functional CEACAM1 family genes, or some derivative thereof,including for example genetic material encoding specific CEACAM1 proteindomains. The genetic material may also contain any cis acting geneticelements that may augment CEACAM1 protein production, including forexample genetic elements that facilitate transcription (gene expression)and/or translation (protein synthesis). The transfer of genetic materialmay be accomplished by any method known in the art.

One exemplar aspect of transplantation includes an act, process, orinstance of transplanting tissue; especially the removal of tissue fromone part of the body or from one individual and its implantation orinsertion in another especially by surgery. The transplantation oftissue can be allogeneic (allograft), which includes transplantation oftissue between genetically different members of the same species. Forexample, nearly all organ and bone marrow transplants are allografts.These may be between brothers and sisters, parents and children, orbetween donors and recipients who are not related to each other. Thetransplantation can also be autologous (autograft), which includestransplantation of an organism's own tissues. A graft or transplantationof tissue from one site to another on the same individual is called anautograft. Autologous transplantation may be used to repair or replacedamaged tissue. For example, autologous bone marrow transplantationpermits the usage of more severe and toxic cancer therapies by replacingbone marrow damaged by the treatment with marrow that was removed andstored prior to treatment. The transplantation of tissue can also besyngeneic, which includes transplantation of tissue between geneticallyidentical members of the same species (e.g., identical twins). Thetransplantation can also be xenogeneic (xenograft), which includestransplantation between members of different species; for example, thetransplantation of animal tissues into humans.

One exemplar characterization of immunological rejection of transplantedtissue includes include those events by which a body's immune systemattacks transplanted or grafted tissue, reacting to them as if they wereharmful. Graft or transplant rejection generally involves thedestruction of the grafted or transplanted tissue by attackinglymphocytes. In clinical transplantation, the types of transplantrejection may be classified into three main types: hyperacute, acute,and chronic.

The present invention also provides materials and/or methods forimparting a tolerogenic state upon engineered tissues. This aspect ofthe present invention can be achieved, at least in part, by impartingupon the engineered tissue CEACAM1 protein function. For example, oneembodiment of the present invention involves the purification of aspecific cell type of interest, followed by a transformation of the cellto produce CEACAM1 protein. These cells are then expanded in cellculture and seeded onto a scaffold of any desirable shape or rigidityprepared from a suitable biomaterial (or biocompatible material, or somecombination) to form a scaffold/biological composite, or tissueengineered construct, that has decrease susceptibility to immunologicalrejection upon transplantation or grafting as replacement tissue.

A further aspect of the present invention provides methods and/ormaterials for imparting a tolerogenic state to tissue afflicted byautoimmune disease, while preserving a body's general immune competence,including for example normal immune responses to pathogens and cancerrisks. This aspect of the present invention may be accomplished, atleast in part, by conveying or imparting CEACAM1 function or activityupon tissue afflicted by autoimmune disease. For example, the presentinvention provides for the targeted transformation of tissue afflictedby autoimmune disease to express CEACAM1 protein. This can beaccomplished, for example, by transfer of genetic information effectivein inducing CEACAM1 protein production directly to tissue afflicted withan autoimmune disease, subsequent to any required exposure of theafflicted tissue. The genetic material that is transferred may include,for example, one or more functional CEACAM1 family genes, or somederivative thereof, including for example genetic material encodingspecific CEACAM1 protein domains. The genetic material may also containany cis acting genetic elements that may augment CEACAM1 proteinproduction, including for example genetic elements that facilitatetranscription (gene expression) and/or translation (protein synthesis).The transfer of genetic material may be accomplished by any method knownin the art, and may be performed subsequent to exposure of the afflictedtissue by surgery, or if surgery is not an option, the effected tissuemay be targeted utilizing receptor-mediated gene transfer technology.

Autoimmune diseases are generally characterized by the body's immuneresponses being directed against its own tissues, causing prolongedinflammation and subsequent tissue destruction. For example, autoimmunedisorders can cause immune-responsive cells to attack the linings of thejoints—resulting in rheumatoid arthritis—or trigger immune cells toattack the insulin-producing islet cells of the pancreas leading toinsulin-dependent diabetes. A healthy immune system recognizes,identifies, remembers, attacks, and destroys bacteria, viruses, fungi,parasites, and cancer cells or any health-damaging agents not normallypresent in the body. A defective immune system, on the other hand,directs antibodies against its own tissues. Any disease in whichcytotoxic cells are directed against self-antigens in the body's tissuesis considered autoimmune in nature. Such diseases include, but are notlimited to, celiac disease, Crohn's disease, pancreatitis, systemiclupus erythematosus, Sjogren's syndrome, Hashimoto's thyroiditis, andother endocrinopathies. Allergies and multiple sclerosis are also theresult of disordered immune functioning.

Examples of different types of viruses used as vectors for the transferof genetic material include, without limitation: retroviruses;adenoviruses; adeno-associated viruses; and herpes simplex viruses.Besides virus-mediated genetic material delivery systems, there areseveral nonviral options for delivery. The simplest method is the directintroduction of the genetic material into target cells. Another nonviralapproach involves the creation of an artificial lipid sphere with anaqueous core. This liposome, which carries the genetic material, iscapable of passing the genetic material through the target cell'smembrane. Genetic material can also get inside target cells bychemically linking the genetic material to a molecule that will bind tospecial cell receptors. Once bound to these receptors, the geneticmaterial constructs are engulfed by the cell membrane and passed intothe interior of the target cell.

Particle mediated transfer of genetic material is also a viable methodto introduce genetic material according to some aspects of the presentinvention. Any method regarding the particle mediated transfer ofgenetic material known in the art may be used. For example, the gene gunis part of a method sometimes called the biolistic (also known asbioballistic) method. Under certain conditions, DNA (or RNA) becomes“sticky,” adhering to biologically inert particles such as metal atoms(usually tungsten or gold). By accelerating this DNA-particle complex ina partial vacuum and placing the target tissue within the accelerationpath, DNA is effectively introduced (Gan, Carol. “Gene Gun AcceleratesDNA-Coated Particles To Transform Intact Cells”. The Scientist; Sep. 18,1989, 3[18]:25. This reference is herein incorporated by reference.).Uncoated metal particles could also be shot through a solutioncontaining DNA surrounding the cell thus picking up the genetic materialand proceeding into the living cell. A perforated plate stops the shellcartridge but allows the slivers of metal to pass through and into theliving cells on the other side. The cells that take up the desired DNA,identified through the use of a marker gene (in plants the use of GUS ismost common), are then cultured to replicate the gene and possiblycloned. The biolistic method is most useful for inserting genes intoplant cells such as pesticide or herbicide resistance. Different methodshave been used to accelerate the particles: these include for examplepneumatic devices; instruments utilizing a mechanical impulse ormacroprojectile; centripetal, magnetic or electrostatic forces; spray orvaccination guns; and apparatus based on acceleration by shock wave,such as electric discharge.

The following invention also provides for the control and/or modulationof a cellular signal transduction pathway(s) designed to transduce andamplify signals emanating from the cell surface and resulting in somecellular effector function. One exemplar characterization of effectorfunction as used herein may include responses resulting in cellulargrowth, differentiation, or the production (and sometimes release ortransport out of the cell) of growth factors and/or other substancesthat have biological activity. For example, the following invention alsoprovides for the stimulation of IL-2 production in a specified cell.This aspect of the invention can be achieved by modifying a specifiedcell to produce a chimeric protein having the ectodomain of the CEACAM1receptor joined to a non-CEACAM1 adaptor portion capable of transducingsignals effective in producing a response resulting in IL-2 production.One exemplar embodiment of the present invention provides chimericconstructs consisting of the extracellular portion of the human CEACAM1protein fused to the transmembrane and cytosolic tail of the mouse zetachain. Generation of BW cells (murine thymoma that lack ab chains ofTCR, but have an intact mIL-2 secretion machinery) transfected withCEACAM1-mouse zeta construct resulted in the production of IL-2 uponaddition of CEACAM1. The engagement of CEACAM1 in these cells activatesthe zeta chain. The BW cells are T cells, which respond to signalsdelivered by the zeta chain by secretion of mIL-2. The amount of mIL-2detected in the medium correlates with CEACAM1 engagement.

Example 1 Residues 43R and 44Q of Carcinoembryonic Antigen Cell AdhesionMolecules-1 (CEACAM1) are Critical in the Protection from Killing byHuman NK Cells

The present inventors have shown that the CEACAM1 (CD66a) homophilicinteractions inhibit the killing activity of NK cells. This novelinhibitory mechanism plays a key role in melanoma immune evasion,inhibition of decidual immune response, and controlling NKautoreactivity in TAP2-deficient patients. These roles are mediatedmainly by homophilic interactions, which are mediated through theN-domain of the CEACAM1. The N-domain of the various members of theCEACAM family shares a high degree of similarity. The present inventorshave addressed which of the CEACAM family members are able to interactwith CEACAM1 and what amino acid residues control this interaction. Inthis section it is shown that CEACAM1 interacts with CEACAM5, but notwith CEACAM6. The present inventors have demonstrated the inability ofCEACAM1 to bind CEACAM6. Importantly, the present inventors provide themolecular basis for CEACAM1 recognition of various CEACAM familymembers. Sequence alignment reveals a dichotomy among the CEACAM familymembers: both CEACAM1 and CEACAM5 contain the R and Q residues inpositions 43 and 44, respectively, whereas CEACAM3 and CEACAM6 containthe S and L residues, respectively. Mutational analysis revealed thatboth ⁴³R and ⁴⁴Q residues are necessary for CEACAM1 interactions. Theinventors have considered the implications for differential expressionof CEACAM family members in tumors.

The inventors in this section directly show that the presence of bothresidues 43R and 44Q in the CEACAM1 is crucial for the homophilicCEACAM1 interaction and that substitution of these residues with the 43Sand 44L residues that are present in CEACAM6 abolishes the inhibitoryeffect. Importantly, the reciprocal substitution of 43S and 44L ofCEACAM6 to the 43R and 44Q residues, respectively, results in the gainof inhibitory heterophilic interactions with the CEACAM1 protein. Thedichotomy of CEACAM family members by recognition of CEACAM1 isdetermined by the presence of R and Q at positions 43 and 44. (GalMarkel et al., The Critical Role of Residues 43R and 44Q ofCarcinoembryonic Antigen Cell Adhesion Molecules-1 in the Protectionfrom Killing by Human NK Cells, The Journal of Immunology, 2004, 173:3732-3739. This reference is herein incorporated by reference.)

Materials and Methods Cells

The cell lines used were the MHC class I-negative 721.221human cellline, the murine thymoma BW cell line that lacks expression of_- and_-chains of the TCR, and the NK tumor line YTS. Primary NK cells wereisolated from PBL using the human NK isolation kit and the autoMACSinstrument (Miltenyi Biotec, Auburn, Calif.). For the enrichment ofCEACAM1-positive NK cells, isolated NK cells were further purified bydepletion of CD16-positive NK cells, using the anti-CD16 mAb B73.1.1 andthe auto MACS instrument. NK cells were grown in culture as previouslydescribed (16). CEACAM1-positive NK clones were identified by flowcytometry using the anti-CEACAM1 mAb 5F4 and were tested for inhibitionin killing assays against .221/CEACAM1 cells.

Antibodies

The Abs used in this work were mAb Kat4c (DakoCytomation, Carpenteria,Calif.), directed against CEACAM1, -5, -6, and -8; the anti-CD99mAb12E7; the rabbit polyclonal anti-CEACAM (DakoCytomation); and thespecific anti-CEACAM1 mAb 5F4 (10). Rabbit polyclonal Abs againstpurified ubiquitin were used as the control.

Generation of CEACAM1-Ig Fusion Protein

The extracellular portion of the CEACAM1 protein was amplified by PCRusing the following primers: the 5′ primer CCCAAGCTTGGGGCCGCCACCATGGGGCACCTCTCAGCC (including the HindIII restriction site) and the3′ primer GCGGATCCCCAGGTGAGAGGC (including the BamHI restriction site).A silent mutation, adenine885guanidine (no change in glycine281) wasperformed by site-directed mutagenesis to cancel the BamHI site in theamplified sequence. The production of the CEACAM1-Ig and CD99-Ig fusionproteins by COS-7 cells and purification on a protein G column werepreviously described (8, 17). The fusion proteins were periodicallyanalyzed for degradation by SDS-PAGE.

Generation of Transfectants

The 721.221 cells expressing CEACAM1 and CEACAM6 proteins were generatedas previously described (7). The CEACAM5 cDNA was subcloned into pcDNA3vector. This construct was permanently transfected to 721.221 cells. Forthe generation of 721.221 cells expressing the CEACAM6 protein fused tothe tail of CEACAM1, the extracellular portion of the CEACAM6 was firstamplified without the GPI-anchoring sequence using the 5′ primerCCCAAGCTTGCCGCCACCATGGGAC CCCCCTCAGCC (including the HindIII restrictionsite) and the 3′ primer AATGGCCCCTCCAGAGACTGTGATCATCGT (including thefirst nine nucleotides of the CEACAM1 transmembrane portion). Thetransmembrane and tail of the CEACAM1 protein were amplified with the 5′primer GTCTCTGGAGGGGCCATTGCTGGCATTG (including the last nine nucleotidesof the CEACAM6 extracellular portion before the GPI anchor motif) andthe 3′ primer GGAATTCCTTACTGCTTTTTTACTTCTGAATA (including the EcoRIrestriction site). Amplified fragments were mixed and fused by anadditional PCR that was performed with the 5′-HindIII primer and the3′-EcoRI primer. The construct was cloned into pcDNA3 vector (InvitrogenLife Technologies, Carlsbad, Calif.) and permanently transfected to721.221 cells. For the generation of BW cells expressing the chimericCEACAM1-_(—) protein, the same technique was used. The extracellularportion of the human CEACAM1 protein was amplified by PCR using the 5′primer CCCAAGCTTGGGGCCGCCACCATGGGGCACCTCTCAGCC (including HindIIIrestriction site) and the 3′ primer GTAGCAGAGAG GTGAGAGGCCATTTTCTTG(including the first nine nucleotides of the mouse _-chain transmembraneportion). The mouse _-chain was amplified by PCR using the 5′ primerCTCTCACCTCTCTGCTACT TGCTAGATGGA (including last nine nucleotides ofhuman CEACAM1 extracellular portion) and the 3′ primer GGAATTCCTTAGCGAGGGGCCAGGGTCTG (including EcoRI restriction site). The two amplifiedfragments were mixed, and PCR was performed with the 5′ HindIII primerand the 3′ EcoRI primer for generation of the CEACAM1-_(—) construct.The CEACAM1-_(—) construct was cloned into pcDNA3 expression vector(Invitrogen Life Technologies) and was stably transfected into BW cells.All transfectants were periodically monitored for expression by stainingwith the appropriate mAb.

Generation of 721.221 Cells Expressing Mutated CEACAM1 or CEACAM6Proteins

For generation of the mutated CEACAM proteins, two overlapping fragmentsof the gene were amplified by PCR. The upstream fragment was amplifiedby using a gene-specific 5′-edge primer (including the HindIIIrestriction site) and an internal 3′ primer bearing the mutation. Thedownstream fragment was amplified using an internal 5′ primer bearingthe mutation and a gene-specific 3′-edge primer (including EcoRIrestriction site). Next, both purified fragments were mixed togetherwith the 5′-edge primer and the 3′-edge primer to generate the mutatedfull-gene cDNA. All different mutants of the same CEACAM gene weregenerated using the same appropriate edge primers and different internalprimers. The various cDNAs were then cloned into the pcDNA3 mammalianexpression vector and stably transfected into the .221 cell line. Alltransfectants were periodically monitored for expression by stainingwith the appropriate mAb. For CEACAM1-RQ43,44SL, the 5_-CEACAM1 edgeprimer was CCCAAGCTTGGGGCCGCCACCATGGGGCACCTCTCAGCC, the 3′-CEACAM1 edgeprimer was GGAATTCCTTACTGCTTTTTTACT TCTGAATA, the 5′ internal primer wasGCCAACAGTCTAATTGTA GGA, and the 3′ internal primer wasTCCTACAATTAGACTGTTGCC. For CEACAM1-R43A, the 5′ internal primer wasGATGGCAACGCTCAAAT TGTA, and the 3′ internal primer wasTACAATTTGAGCGTTGCCATC. For CEACAM1-Q44L, the 5′ internal primer wasATGGCAACCGTCTA ATTGTAG, and the 3′ internal primer wasCTACAATTAGACGGTTGC CAT. For CEACAM6-SL43,44RQ, the 5′-CEACAM6 edgeprimer was CCCAAGCTTGCCGCCACCATGGGACCCCCCTCAGCC, the 3′-CEACAM6 edgeprimer was GGAATTCCCTATATCAGAGCCACCCTGG, the 5′ internal primer wasGGCAACCGTCAAATTGTAGGA, and the 3′ internal primer wasTCCTACAATTTGACGGTTGCC. For CEACAM6-S43R, the 5′ internal primer wasGATGGCAACCGTCTAAT TGTA, and the 3′ internal primer wasTACAATTAGACGGTTGCCATC. For CEACAM6-L44Q, the 5′ internal primer was GATGGCAACAGTCAAATTGTA, and the 3′ internal primer was TACAATTTGACTGTTGCCATC.

Cytotoxicity Assays

The cytotoxic activity of YTS and NK cells against various targets wasassayed in 5-h [35S]Met release assays, as described previously (16). Inexperiments in which rabbit polyclonal Abs were included, the finalconcentration was 20 _g/ml. In all cytotoxicity assays performed,spontaneous release did not exceed 20% of maximal labeling.

Cross-Linking of BW/CEACAM1 Cells

BW/CEACAM1 cells (0.5_105/well) were incubated with various amounts ofKat4c mAb on ice for 1 h in 96-well, round-bottom microplates (NalgeNunc, Rochester, N.Y.). Treated BW/CEACAM1-_ cells, present in 200 _l ofRPMI 1640 complete medium, were then cultured in 96-well, flat-bottommicroplates (Nalge Nunc) precoated with 1 _g/well sheep anti-mouse IgGAbs (ICN Biomedicals, Costa Mesa, Calif.) for 24 h at 37° C. Supernatantwas harvested, and the amount of murine IL-2 (mIL-2) was determined byELISA.

CEACAM1 Protein does not Recognize CEACAM6

The surface expression of the CEACAM1 protein on tumors is associatedwith poor prognosis in melanoma and lung adenocarcinoma patients.Moreover, the CEACAM1 homophilic interactions confer protection fromhuman NK-mediated cytotoxicity, and in some melanoma patients bearingCEACAM1-positive tumors, a dramatic increase in the proportion ofCEACAM1-positive NK cells was observed. Because heterophilicinteractions of the CEACAM1 protein with the CEACAM6 protein werereported previously (12), and because some CEACAM1-positive tumorsdown-regulate the CEACAM1 protein expression and instead replace it withCEACAM6 (14, 15), it was investigated whether CEACAM1 can interact withCEACAM6.

721.221 (.221) cells were transfected with the CEACAM1 cDNA(.221/CEACAM1) and with the CEACAM6 cDNA (.221/CEACAM6) as described inMaterials and Methods. The expression level was monitored with the Kat4cmAb (FIG. 1). For measuring direct binding of CEACAM1 to the transfectedcells, the extracellular portion of the CEACAM1 fused to the Fc portionof human IgG1 (CEACAM1-Ig) was used in flow cytometry binding assays.The production and purification of the CEACAM1-Ig fusion protein wereperformed as described in Materials and Methods. Homophilic binding ofthe CEACAM1-Ig fusion protein was observed only in the .221/CEACAM1cells (FIG. 1). In contrast, despite a slightly higher expression levelof the CEACAM6 protein (detected by Kat4c mAb, FIG. 1), CEACAM1-Ig didnot bind to .221/CEACAM6 cells (FIG. 1). The control CD99-Ig fusionprotein did not stain any of the transfectants.

The potential heterophilic interactions between the CEACAM1 and CEACAM6proteins were further investigated using the BW cell system. BW cellswere stably transfected with the extracellular portion of the CEACAM1fused to mouse _-chain (BW/CEACAM1-_) as described in Materials andMethods. The specific functionality of BW/CEACAM1-_ was assessed bycrosslinking the CEACAM1 receptor using different amounts of immobilizedKat4c mAb as described in Materials and Methods. Engagement of theCEACAM1 protein elicited the synthesis and secretion of mIL-2 in a dosedependent manner (FIG. 2A). Treatment with the control anti-CD99 12E7gave no response (FIG. 2A). Next, BW parental cells and BW/CEACAM1-_were co cultured with irradiated .221, 0.221/CEACAM1, or .221/CEACAM6cells for 48 h. Significant amounts of mIL-2 were detected in thesupernatant of BW/CEACAM1-_ cells co incubated with .221/CEACAM1 cells(FIG. 2B). In contrast, no mIL-2 was detected when BW/CEACAM1-_ cellswere co incubated with .221 or .221/CEACAM6 cells (FIG. 2B). Nosecretion of mIL-2 was observed when parental BW cells were used (FIG.2B). These combined results suggest that the CEACAM1 and CEACAM6proteins do not bind or functionally interact.

GPI Anchorage of CEACAM6 is not Responsible for the Lack of HeterophilicInteractions with the CEACAM1

Several explanations may account for the potential lack of heterophilicinteractions between CEACAM1 and CEACAM6 proteins. CEACAM1 is atransmembrane protein, whereas CEACAM6 is GPI-anchored to the cellmembrane. It is possible that the GPI anchor of CEACAM6 and the absenceof transmembrane and cytosolic portions weaken the interaction.Furthermore, it is possible that other transmembrane elements play a keyrole in the interactions. For example, a cysteine residue located in thetransmembrane domain of HLA-C was reported to be crucial for theinhibition mediated by an unknown inhibitory NK receptor. To testwhether the GPI anchor of CEACAM6 protein is responsible for the lack ofCEACAM1 binding, a chimeric construct comprised of the entireextracellular portion of CEACAM6 fused to the transmembrane and tailportions of CEACAM1 (CCM6-TailCCM1) was generated. The .221 cells werestably transfected with the CCM6-TailCCM1 construct(.221/CCM6-TailCCM1). The expression level of the CCM6-TailCCM1 chimericprotein, detected by Kat4c mAb, was similar to that of the other.221/CEACAM stable transfectants (FIGS. 1 and 3A). Importantly, nobinding of CEACAM1-Ig was observed to .221/CCM6-TailCCM1cells (FIG. 3A).In agreement with the binding results, the presence of mIL-2 was notdetected in the supernatant of BW/CEACAM1 cells co incubated with.221/CCM6-TailCCM1cells (FIG. 3B). These results suggest that the lackof heterophilic interactions of CEACAM1 and CEACAM6 may not be due tothe transmembrane and cytosolic tail portions of the proteins.

Residues 43R and 44Q are Critical for CEACAM1 Binding

CEACAM-related proteins share a common basic structure of severalsequential Ig-like domains. The Ig-like domains serve as fundamentalbuilding blocks of the various CEACAM-related proteins, and they differonly slightly from one protein to another. Importantly, the binding siteof the CEACAM1 is located in the N-domain (13). Sequence alignment ofthe N-domains of CEACAM-related proteins, including CEACAM1, CEACAM3,CEACAM5, and CEACAM6, revealed exceptional homology (FIG. 4). Within theCEACAM1 N-domain, several amino acid residues may be crucial forbinding. These include amino acids 39V and 40D (13) and the salt bridgebetween 64R and 82D (13). All of the above-reported amino acid residuesare present in the N-domain of CEACAM6 (FIG. 4), implying that they maynot account for the lack of heterophilic interactions with the CEACAM1protein.

Three short sequences located in the N-domain of CEACAM5 are criticalfor CEACAM5 homophilic interactions (Taheri et al. (20)). These shortsequences include 30GYSWYK, 42NRQII, and 80QNDTG. Importantly, thesethree short sequences are present in the N-domain of the CEACAM1protein. However, only the 30GYSWYK and 80QNDTG short sequences arepreserved in the N-domain of the CEACAM6 protein, whereas the 43R44Qresidues are replaced with 43S44L residues within the 42NRQII sequence(FIG. 4). A mutated construct was generated of the CEACAM6 gene thatincludes amino acids R and Q at positions 43 and 44 instead of S and L,respectively (CCM6-SL43,44RQ). In addition, the reciprocal mutation inCEACAM1 was generated that includes amino acids S and L at positions 43and 44 instead of R and Q, respectively (CCM1-RQ43,44SL). The .221 cellswere stably transfected with the various constructs and tested forexpression using Kat4c mAb (FIG. 5A). The expression levels of themutated proteins were similar to those of CEACAM1 and CEACAM6 (FIGS. 1and 5A).

Next, .221/CCM6-SL43,44RQ and .221/CCM1-RQ43,44SL were tested in flowcytometry binding assays using CEACAM1-Ig. Remarkably, substitution of43R44Q with 43S44L in .221/CCM1-RQ43,44SL abolished homophilic binding(FIG. 5A). This abolishment was probably not merely due to a stericdisturbance of the CEACAM1 N-domain structure, because the reciprocalmutation, 43S44L with 43R44Q in .221/CCM6-SL43,44RQ, conferred strongbinding of the CEACAM1-Ig fusion protein (FIG. 5A). The CD99-Ig fusionprotein did not stain any of the transfectants. Strikingly, recognitionof the mutated CEACAM1 protein by the conformation-dependentanti-CEACAM1 mAb 5F4 (10, 13), was abolished, whereas specific stainingof .221/CCM6-SL43,44RQ was observed (FIG. 5B). These results imply thatthe 43R and 44Q residues are critically involved in conferring theappropriate conformation required for recognition by CEACAM1.

The binding results were also confirmed by functional assays using theBW cell system. Significant amounts of mIL-2 were detected only insupernatants of BW/CEACAM1-_ cells co incubated with irradiated.221/CCM6-SL43,44RQ or .221/CEACAM1 cells (FIG. 5C). The stronger mIL-2induction after incubation of BW/CEACAM1-_ cells with the.221/CCM6-SL43,44RQ cells compared with .221/CEACAM1 cells might be dueto the higher protein expression measured by Kat4c mAb (FIGS. 1 and 5A).The presence of mIL-2 could not be detected in the supernatants ofBW/CEACAM1-_ cells co incubated with irradiated .221/CEACAM6 cell or.221/CEACAM1 RQ43,44SL (FIG. 5C). Mouse IL-2 was not detected when BWparental cells were used. These results show that residues 43R44Q arecritical for the functional CEACAM1 interactions.

Residues 43R and 44Q are Critical for CEACAM1-Mediated Inhibition of NkCell Cytotoxicity

CEACAM1 plays a major role in regulation of NK cell cytotoxicity (7),inhibition of decidual immune responses after activation (8), andconferring protection from NK autoreactivity in TAP2-deficient patients(9). To test whether residues 43R44Q would also be important in theinhibition of NK killing, NK cells from several healthy donors wereisolated, depleted the CD16-positive NK cells, activated NK clones werethen cultured as described in Materials and Methods, and stained forCEACAM1 expression.

CEACAM1-positive NK clones were assayed for cytotoxic activity against.221 parental cells and various stable transfectants, including221/CEACAM1, 0.221/CEACAM6, 0.221/CCM6-Tail-CCM1, 0.221/CCM1-RQ43,44SL,and .221/CCM6-SL43,44RQ cells. NK cytotoxicity assays were performedwith no Ab included, in the presence of anti CEACAM polyclonal Abs, orwith the control anti-ubiquitin polyclonal Abs. All NK clonesefficiently killed parental .221 cells regardless of whether Abs wereincluded (representative clone NK23 is presented in FIG. 6). Aspreviously reported (7-9), inhibition of NK killing was observed when.221/CEACAM1 cells were used. This inhibition was the result of CEACAM1inhibition, because anti-CEACAM Abs abrogated this effect (FIG. 6). Thelack of heterophilic interactions between CEACAM1 and CEACAM6 wasevident in the NK killing assays, because .221/CEACAM6 and.221/CCM6-TailCCM1 cells were killed as efficiently as parental .221cells (FIG. 6). In agreement with the above results (FIG. 5), noinhibition was observed when .221/CCM1 RQ43,44SL cells were used (FIG.6). Remarkably, a strong inhibition of killing was observed whenCCM6-SL43,44RQ cells were used as targets (FIG. 6). The inhibition waseven stronger than that observed with the homophilic CEACAM1interactions, probably due to the higher CCM6-SL43,44RQ expression. Thisinhibition was the result of heterophilic interactions with CEACAM1protein on NK cells, because killing was restored when anti-CEACAM Abswere included in the assay (FIG. 6). The control anti-ubiquitin hadlittle or no effect when included in the assays (FIG. 6).

Specificity of CEACAM1 Binding to CEACAM6 is Controlled by the Presenceof Both 43R and 44Q Residues

To determine whether both residues are required for binding, the aminoacid residues in positions 43 and 44 in CEACAM1 (contains 43R44Q) andCEACAM6 (contains 43S44L) were mutated. Using site-directed mutagenesisin the CEACAM1, the 43R residue was changed to 43A (CCM1-R43A) and the44Q residue was changed to 44L (CCM1-Q44L). In CEACAM6, the 43S waschanged to 43R (CCM6-S43R) and 44L was changed to 44Q (CCM6-L44Q). Allmutants were generated as described in Materials and Methods and stablytransfected into .221 cells. The expression level was monitored by Kat4cmAb, and conformation was monitored by 5F4 mAb (FIG. 7A). Importantly,substitution for 44Q in CEACAM1 protein by 44L in .221/CCM1-Q44Lcompletely abrogated 5F4 binding, whereas the Kat4c binding observed wassimilar to that of wild type CEACAM1 (FIG. 7A). This suggests that the44Q residue is essential for maintaining appropriate conformation, whichis crucial for binding of 5F4 mAb. Indeed, this mutation also resultedin a lack of recognition by the CEACAM1-Ig (FIG. 7B). Similar resultswere obtained when both 44Q and 43R residues in CEACAM1 were mutated(FIG. 5). The reciprocal mutant .221/CCM6-L44Q was not recognized by 5F4mAb, suggesting that it is not the only factor crucial for conferringthe appropriate conformation for 5F4 (FIG. 7A). Compatible with thelatter observation, no binding of CEACAM1-Ig to .221/CCM6-L44Q could bedetected (FIG. 7B). Point mutation in the 43R residue of CEACAM1 did notaffect 5F4 mAb binding (FIG. 7A), suggesting that by itself the 43Rresidue had no significant effect on conformation of 5F4-recognizedepitope. Despite that, the CEACAM1-Ig fusion protein did not recognize.221/CEACAM1-R43A cells (FIG. 7B). Elements of CEACAM1 other than thepresence of the 5F4 epitope and the presence of the 44Q residue may playa crucial role in CEACAM1 binding. In this regard, it should be notedthat the expression level of .221/CCM1-R43A obtained was lower than theexpression levels of the other transfectants (FIG. 7A), which mightaccount for the lack of efficient binding of CEACAM1-Ig. Therefore, totest whether the 43R residue by itself can confer CEACAM1 binding, the43S of CEACAM6 was replaced with 43R. The .221/CCM6-S43R cells were notstained by either the 5F4 mAb (FIG. 7A) or the CEACAM1-Ig fusion protein(FIG. 7B). Gain-of-binding of CEACAM1-Ig to CEACAM6 was evident onlywhen both 43S and 44L residues were replaced with 43R and 44Q,respectively (FIG. 5A). Thus, both 43R and 44Q residues are critical forinteraction with CEACAM1.

These binding results were also confirmed in functional killing assays.To optimize the isolation of the experimental variables, the YTS NKtumor line was used. The NK tumor line YTS was either mock-transfected(YTS/control) or transfected with CEACAM1 protein (YTS/CCM1) aspreviously described (7) and tested in killing assays against thevarious .221 transfectants. The function of CEACAM1 protein in YTS/CCM1cells was confirmed, because killing of .221/CEACAM1 cells was inhibitedcompared with killing by YTS/control cells, whereas .221/CEACAM6 and.221/CCM6-TailCCM1 cells were killed with similar efficiency (FIG. 7C).In agreement with the CEACAM1-Ig binding results, the inhibition ofYTS/CCM1 cells was abolished when the .221/CCM1-RQ43,44SL and.221/CCM1-Q44L transfectants were used as targets (FIG. 7C),demonstrating the critical role of residue 44Q. In agreement with theabove observation, the presence of 44Q only is not enough to conferinhibition, and only a mild inhibitory effect was observed when the.221/CCM1-R43A cells were used (FIG. 7C). This result was also supportedby the observation that inhibition of YTS/CCM1 cells by heterophilicinteractions with CEACAM6 was observed only with the .221/CCM6-SL43,44RQdouble mutation, whereas no inhibition was observed when .221/CCM6-S43Ror .221/CCM6-L44Q cells were used (FIG. 7C). Similar results wereobtained with primary NK clones. Both R and Q residues in positions 43and 44, respectively, are required for functional interaction withCEACAM1.

CEACAM1 can heterophilically interact with the CEACAM5 protein (12). TheCEACAM5 protein is the only CEACAM family member other than CEACAM1 thatcontains 43R44Q residues (FIG. 4). The interactions between CEACAM1 andCEACAM5 was also examined. The expression level of .221/CEACAM5transfectant was monitored with Kat4c and was similar to that of theother CEACAM transfectants (FIG. 7D). The .221/CEACAM5 cells were notstained by the anti-CEACAM1-specific 5F4 mAb (FIG. 7D). Efficientheterophilic binding of the CEACAM1-Ig fusion protein to .221/CEACAM5was observed (FIG. 7D).

Modulation of CEACAM1 Activity in Adoptive Immunotherapy

Adoptive immunotherapy is a general term describing the transfer ofimmunocompetent cells (i.e. lymphocytes) to a patient for the treatmentof a disease, such as cancer. For example, a cancer patient's immunesystem is sometimes capable of delaying tumor progression and on rareoccasions can eliminate the tumor altogether. A variety of immunologictherapies designed to stimulate the patient's own immune system exist.For example, passive non-specific immunotherapy might involve thetransfer of lymphokine activated killer cells. Another example ispassive specific immunotherapy, including the transfer of specificimmune cells such as cytotoxic T-lymphocytes or lymphocytes producingspecific antibodies.

One example of adoptive immunotherapy involves removing lymphocytes fromthe patient, boosting their anti-cancer activity, growing them in largenumbers, and then returning them to the patient. For example, strongerresponse against tumor cells is obtained using lymphocytes isolated fromthe tumor itself. These tumor-infiltrating lymphocytes (TILs) are grownin the presence of IL-2 and returned to the body to attack the tumor.Researchers are also using radiolabeled monoclonal antibodies for tumorantigens to even more closely identify lymphocytes specific for tumorcells.

One object of the present invention provides materials and/or forenhancing the efficacy of Tumor Infiltrating Lymphocyte based therapy inthe treatment of cancer, which includes the modulation of CEACAM1function in a population of Tumor Infiltrating Lymphocytes. The methodmay involve, for example, the disruption of a CEACAM1 protein-proteininteraction, that may be either homotypic or heterotypic. The method mayalso involve, for example, the negative modulation of CEACAM1 geneexpression and/or translational efficiency in a population of TumorInfiltrating Lymphocytes.

Strategies and Protocals for TIL Isolation, Expansion and Treatment

One major challenge in adoptive immunotherapy is to develop immune cellswith specific antitumor reactivity that could be generated in largeenough quantities for transfer to tumor bearing patients. Thelymphocytes infiltrating a tumor (TILs) are both cytotoxic and helper Tcells and have specific antitumor activity, presumably because theyrecognize specific tumor antigens. TIL therapy involves harvesting thetumor-infiltrating lymphocytes from the tumor itself and then isolatingthe cells by growing single cell suspensions from the tumor. Afterseveral weeks of culture in the presence of IL-2, the activated TILcells are transfused back into the patient [Rosenberg S A, Lotze M T,Yang J C et al. Prospective randomized trial of high-dose interleukin-2alone or in conjunction with lymphokine-activated killer cells for thetreatment of patients with advanced cancer. J Nat Cancer Inst 1993;85:622-32. This reference is herein incorporated by reference.] Thistechnique may require an additional biopsy procedure for the solepurpose of harvesting a portion of tumor for subsequent isolation of theTILs.

When cultured in the presence of IL-2, TILs can be activated andexpanded in great numbers. TILs can be prepared from primary ormetastatic tumors. The specimens are excised and digested in an enzymesolution, and the sterile, single-cell suspension is incubated in thepresence of IL-2. In three to four weeks, an activated T-lymphocytepopulation is generated, and approximately 1011 cells are reinfused intothe patient together with IL-2. The lymphocyte subpopulations varyaccording to the histology of the original tumor, culture conditions,IL-2 concentration, and other variables. The expansion of HumanTumor-Infiltrating Lymphocytes has been characterized under differentconditions. There are many strategies and protocols for TIL isolation,expansion and treatment, including for example, the followingreferences, which are herein incorporated by reference:

-   [Yannelli J R, Wroblewski J M., On the road to a tumor cell vaccine:    20 years of cellular immunotherapy., Vaccine. 2004 Nov. 15;    23(1):97-113. This reference is herein incorporated by reference.]-   [Yamaguchi Y et al., Adoptive immunotherapy of cancer using    activated autologous lymphocytes—current status and new strategies.,    Hum Cell. 2003 December; 16(4):183-9. This reference is herein    incorporated by reference.]-   [Colin C. Malone et al. Characterization of Human Tumor-Infiltrating    Lymphocytes Expanded in Hollow-Fiber Bioreactors for Immunotherapy    of Cancer. Cancer Biotherapy & Radiopharmaceuticals. October 2001,    Vol. 16, No. 5: 381-390. This reference is herein incorporated by    reference.]-   [Whiteside T L, Miescher S, Hurlimann J, Moretta L, von Fliedner V.    Separation, phenotyping and limiting dilution of T lymphocytes    infiltrating human solid tumors. Int J Cancer 1986; 37:803-11. renal    cell carcinoma. J Urol 1993; 150:1384-90. This reference is herein    incorporated by reference.]-   [Rosenberg S A, Speiss P, Lafreniere R. A new approach to adoptive    immunotherapy of cancer with tumor-infiltrating lymphocytes. Science    1986; 233:1318-21. This reference is herein incorporated by    reference.]-   [Knazek R A, Wu Y W, Aebersold P A, Rosenbeg S A. Culture of tumor    infiltrating lymphocytes in hollow fiber bioreactors. J Immunol    Methods 1990; 127:29-37. This reference is herein incorporated by    reference.]-   [Bukowski R M, Sharfman W, Murthy S, et al. Clinical results and    characterization of tumor-infiltrating lymphocytes with or without    recombinant interleukin-2 in human metastatic renal cell carcinoma.    Cancer Res 1991; 51:4199-4205. This reference is herein incorporated    by reference.]-   [Lewko W M, Good R W, Bowman D, Smith T K, Oldham R K. Growth of    tumor derived activated T-cells for the treatment of cancer. Cancer    Biother 1994; 9:211-24. This reference is herein incorporated by    reference.]-   [Hillman G G, Wolf M L, Montecillo E, Younes E, Ali E, Pontes J E,    Haas G P. Expansion of activated lymphocytes obtained from renal    cell carcinoma in an automated hollow fiber bioreactor. Cell    Transplant 1994; 3:263-271. This reference is herein incorporated by    reference.]-   [Yannelli J R, Hyatt C, McConnell, et al. Growth of    tumor-infiltrating lymphocytes from human solid cancers: summary of    a 5-year experience. Int J Cancer 1996; 65: 413-22. This reference    is herein incorporated by reference.]-   [Schiltz P M, Beutel L D, Nayak S K, Dillman R O. Characterization    of tumor infiltrating lymphocytes derived from human tumors for use    as adoptive immunotherapy of cancer. J Immunother 1997; 20:377-386.    This reference is herein incorporated by reference.]-   [Topalian S L, Solomon D, Avis F P, et al. Immunotherapy of patients    with advanced cancer using tumor-infiltrating lymphocytes with    recombinant interleukin-2: a pilot study. J Clin Oncol 1988;    6:839-53. This reference is herein incorporated by reference.]-   [Rosenberg S A, Packard B S, Aebersold P M, et al. Use of tumor    infiltrating lymphocytes and interleukin-2 in the immunotherapy of    patients with metastatic melanoma: a preliminary report. N Engl J    Med 1988; 319: 1676-80. This reference is herein incorporated by    reference.]-   [Kradin R L, Kurnick J T, Lazarus D S, et al. Tumor-infiltrating    lymphocytes and interleukin-2 in treatment of advanced cancer.    Lancet 1989; 18:577-80. This reference is herein incorporated by    reference.]-   [Dillman R O, Oldham R K, Barth N M, et al. Continuous interleukin-2    and tumor-infiltrating lymphocytes as treatment of advanced    melanoma; a National Biotherapy Study Group trial. Cancer 1991;    68:1-8. This reference is herein incorporated by reference.]-   [Oldham R K, Dillman R O, Yannelli J R, et al. Continuous infusion    interleukin-2 and tumor-derived activated cell as treatment of    advanced solid tumors; a National Biotherapy Study Group. Molec    Biother 1991; 3:68-73. This reference is herein incorporated by    reference.]-   [Belldegrun A, Pierce W, Kaboo R, et al. Interferon-a primed    tumor-infiltrating lymphocytes combined with interleukin-2 and    interferon-a as therapy for metastatic renal cell carcinoma. J Urol    1993; 150:1384-90. This reference is herein incorporated by    reference.]-   [Rosenberg S A, Yannelli J R, Yang J C, et al. Treatment of patients    with metastatic melanoma with autologous tumor infiltrating    lymphocytes and interleukin-2. J Natl Cancer Inst 1994; 86:1159-66.    This reference is herein incorporated by reference.]-   [Goedegebuure P S, Douville L M, Li H, et al. Adoptive immunotherapy    with tumor-infiltrating lymphocytes and interleukin-2 in patients    with metastatic malignant melanoma and renal cell carcinoma: a pilot    study. J Clin Oncol 1995; 13:1939-49.]-   [Fuji K, Karachi H, Takakuwa K, et al. Prolonged disease-free period    in patients with advanced epithelial ovarian cancer after adoptive    transfer of tumor-infiltrating lymphocytes. Clin Cancer Res 1995;    1:501-7.]-   [Queirolo P, Ponte M, Gipponi M, et al. Adoptive immunotherapy with    tumor infiltrating lymphocytes and subcutaneous recombinant    interleukin-2 plus interferon alfa-2a for melanoma patients with    nonresectable distant disease: a phase I/II pilot trial. Ann Surg    Oncol 1999; 6:272-278.]-   [Semino C, Martini L, Queirolo P, et al. Adoptive immunotherapy of    advanced solid tumors: an eight year clinical experience. Anticancer    Research 1999; 19: 5645-5650.]

Example 2 Biological Function of the Soluble CEACAM1 Protein andImplications in TAP2-Deficient Patients

Interactions of natural killer (NK) cells with MHC class I proteinsprovide the main inhibitory signals controlling NK killing activity.However, TAP2-deficient patients suffer from autoimmune manifestationsonly occasionally in later stages of life. The present inventors havedemonstrated that the CEACAM1-mediated inhibitory mechanism of NKcytotoxicity plays a major role in controlling NK autoreactivity inthree newly identified TAP2-deficient siblings. This novel mechanismprobably compensates for the lack of MHC class I mediated inhibition.The CEACAM1 protein can also be present in a soluble form and thebiological function of the soluble form of CEACAM1 with regard to NKcells was investigated by the present inventors. In this section, thepresent inventors will show that the homophilic CEACAM1 interactions areabrogated in the presence of soluble CEACAM1 protein in a dose-dependentmanner. Importantly, the amounts of soluble CEACAM1 protein detected insera derived from the TAP2-deficient patients were dramatically reducedas compared to healthy controls. This dramatic reduction does not dependon the membrane-bound metalloproteinase activity. The present inventorsdemonstrated that the expression of CEACAM1 and the absence of solubleCEACAM1 observed in the TAP2-deficient patients practically maximize theinhibitory effect and probably help to minimize autoimmunity in thesepatients.

In this section, the present inventors will show that the solubleCEACAM1 protein blocks the CEACAM1-mediated inhibition of NK cellkilling activity in a dose-dependent manner. Moreover, the presentinventors will demonstrate that serum CEACAM1 levels among theTAP2-deficient patients are decreased when compared to normalindividuals, in agreement with the dominant role of the CEACAM1-mediatedinhibition in controlling NK autoreactivity in TAP2-deficient patients.(Gal Markel et al., Biological function of the soluble CEACAM1 proteinand implications in TAP2-deficient patients, Eur. J. Immunol. 2004. 34:2138-2148. This reference is herein now incorporated by reference.)

Materials and Methods Cells

The cell lines used in this work were 721.221 (.221) cells and .221cells stably transfected with the CEACAM1 protein (.221/CEACAM1) [17].Primary human NK cells were isolated and cultures maintained asdescribed [30]. An Institutional Review Board approved these studies andinformed consent was provided according to the Declaration of Helsinki.

Antibodies and Fusion Proteins

The following mAb were used: anti-CEACAM1, 5, 6, 8 mAb Kat4c (DAKO),anti-NKp46 mAb 461-G1 [9, 20] and pan anti-MHC class I mAb W6/32. Inaddition, several fluorochrome conjugated mAb were used, including theanti-CD3-CyChrome (clone HIT3a, PharMingen), anti-CD4-FITC (clone MT310,DAKO), anti-CD8-PE (clone DK25, DAKO), anti-CD16-Biotin (clone LNK16,Serotec), anti-CD56-PE (clone B159, PharMingen) and the Kat4c-FITC(DAKO). Polyclonal rabbit anti-human CEACAM (DAKO) antibodies were usedfor blocking in killing assays and the rabbit anti-ubiquitin antibodieswere used as control. The production and purification of the LIR1-Ig[48], KIR2DL2-Ig [49], CEACAM1-Ig [19] and CD99-Ig [7] were performed asdescribed [19].

Flow Cytometry

Multiple staining analyses of PBL were performed with the followingfluorochrome-conjugated antibodies: anti-CD3-CyChrome, anti-CD16-Biotinfollowed by streptavidin-Cy5 (Jackson ImmunoResearch), anti-CD56-PE andanti-CEACAM-FITC. Another set of antibodies used, included theanti-CD3-CyChrome, anti-CD4-FITC and the anti-CD8-PE. Cells werepretreated with 20% human serum to block nonspecific binding and controlantibodies matching in isotype as well as in the fluorochrome were usedas background. Staining with the various Ig-fusion proteins wasperformed as previously described [17, 19].

Detection of Serum CEACAM1 Level by ELISA

A standard sandwich ELISA protocol was used to quantify the amount ofsoluble CEACAM1 protein in the serum. The specific anti-CEACAM1 5F4 mAbwas used as capturing antibody. For detection, biotinylated Kat4c mAbwas used, followed by streptavidin-horseradish peroxidase (JacksonImmunoResearch). Biotinylation of the Kat4c mAb was performed withSulfo-NHS-SS-Biotin (Pierce) according to the manufacturer'sinstructions. The quantification was calculated according to standardsamples of CEACAM1-Ig fusion proteins.

Killing Assays

The cytotoxic activity of NK cells against the various targets wasassayed in 5-h 35S-release assays, as described [17]. In experimentswhere antibodies were included, the final Ab concentration was 20 ug/ml.In all assays performed, the spontaneous release did not exceed 25% ofthe maximal labeling.

MBMP Assays

Cells were tested for surface expression of various proteins followingactivation of MBMP with PMA [34-36]. Tested cells were distributed at5×104 cells/well in 96-well U-bottom plates in 200 ul RPMI 1640 (Sigma)supplemented with 10% heat-inactivated FCS. When PMA was included thefinal concentration was 4 ng/ml. The final concentration of themetalloproteinase inhibitor BB-94 was 2 uM. The cells were incubated ina 5% CO2 humidified incubator at 37° C. for 2 hours, washed twice andanalyzed by FACS.

Characterization of PBL Subpopulations in TAP2-Deficient Patients

Three new siblings were identified that suffer from a deficiency in theTAP2 subunit [20]. This deficiency is inherited in an autosomalrecessive pattern (FIG. 8). The siblings, patients A, B and C(19-year-old female, 14 and 9 years old males, respectively) displayedseveral clinical manifestations, similar to those displayed by otherTAP2-deficient patients [24-26]. The other five sisters, as well as theparents, who are first cousins, showed no clinical symptoms and areconsidered healthy (FIG. 8).

PBL were obtained from the three patients, as well as from a healthysister. All three patients had normal values of lymphocytes among theirperipheral blood. The cells were stained for CD56 and CD3 expression todifferentiate between various lymphocyte subpopulations, including NKcells (CD56+ CD3−), T cells (CD56− CD3+) and NKT cells (CD56+ CD3+).Normal distribution of these subpopulations could be observed among thethree patients (FIG. 9A), implying that even the low level of MHC classI expression observed in the patients [20] is still sufficient to selectfor proper development of lymphocyte subpopulations.

Expression analysis of various MHC class I recognizing NK inhibitoryreceptors on NK cells obtained from the patients revealed marked changes[20]. Therefore, the expression pattern of the CD16 and the CD56 on thefreshly isolated NK cells were further characterized. Patient Aexhibited an increase in the percentage of CD16− subpopulation (22%) ascompared to the healthy sister (5%) (FIG. 9B), whereas patients B and Cdisplayed a milder trend (8% and 11%, respectively) (FIG. 9B). Moreover,double staining showed a skewing in the different subpopulations betweenthe three patients and the healthy sister; the CD56dim CD16− subset wasincreased in patients A (7%) and C (9%) compared to the healthy sister(3%) (FIG. 9B), the CD56bright CD16− subset was markedly increased inpatients A (15%) and B (6%) compared to the healthy sister (2%) (FIG.9B) and the CD56bright CD16+ subset was increased only in patient A (8%)compared to the healthy sister (1%) (FIG. 9B). The NKp46 expression onNK cells was impaired in all patients as compared to the healthy sister,most prominently in patient A (Table 1). A remarkable difference in theCD4/CD8 ratio was observed among the T cell subpopulations; 65% of the Tcells from the healthy sister expressed the CD4 receptor and 35% theCD8. There were no double-positive CD4+CD8+ T cells. In contrast, CD4was detected on the surface of 91%, 92% and 87% of the T cells analyzedfrom patients A, B and C, respectively (Table 1). CD8 was detected onthe surface of 9%, 8% and 13% of the T cells analyzed from patients A, Band C, respectively (Table 1). A similar skew in the CD4/CD8 ratio wasalso observed among T cells in other TAP2-deficient patients [24].

TABLE 1 Receptor expression^(a)) Sub- Subject population CD4 CD8 NKp46Patient A NK — — <5%  T 91%  9% 0% Patient B NK — — 60%  T 92%  8% 0%Patient C NK — — 20%  T 87% 13% 0% Sister NK — — 100%  T 68% 35% 0%^(a))Percentages of the expression of the indicated receptors on NKcells derived from patients A, B, C and their healthy sister.

Loss of MHC Class I Mediated Inhibition in TAP2-Deficient Patients

Polyclonal EBV-transformed B cell lines were generated from patients A,B and C (EBV-A, -B and -C, respectively) as well as from the healthymother (EBV-M). These cells were stained for MHC class I expressionusing W6/32 mAb. A 30-fold reduction in the expression of MHC class Iproteins was observed on EBV-A, -B and -C as compared to EBV-M (FIG.10). Nevertheless, despite the deficiency in the TAP2 subunit, some MHCclass I alleles were still expressed on cell surface (FIG. 10). The MHChaplotype of all patients is HLA-A*03, B*07, Bw6, Cw*07, DRB1*15, DRB5and DQB1*06. The low expression of MHC class I proteins in the patients'cells is allele specific, as no expression was observed when an mAbspecific for HLA-A*03 was used [20]. Next, it was tested whether the lowlevels of MHC class I proteins are still sufficient for interactionswith inhibitory NK receptors. EBV-A, -B, -C and -M cells were stainedwith the KIR2DL2-Ig, recognizing HLA-Cw7 [27] that is present on thepatients' cells, and with the LIR1-Ig that recognizes a broad spectrumof HLA proteins [28]. As expected, EBV-M cells were efficiently stainedby both the KIR2DL2-Ig and LIR1-Ig (FIG. 10). In contrast, little or nostaining was observed on EBV-A, -B or -C cells (FIG. 10).

Unusual CEACAM1 Expression on NK Cells Derived from TAP2-DeficientPatients

Fresh NK cells derived from the three TAP2-deficient patients as well asfrom the healthy sister were negative for CEACAM1 expression [20]. Thepurified NK cells were next activated with IL-2 and grown either as bulkcultures or as NK clones. CEACAM1 expression was monitored using the 5F4mAb [29]. As reported [17, 19], around 90% of the NK clones obtainedfrom the healthy sister or the mother did not express CEACAM1 (FIG. 11).Strikingly, all of the NK clones (100%) obtained from patient Aexpressed the CEACAM1 in unusually high levels (30-fold abovebackground). (see for example NK clone 2 in the healthy sister in FIG.11, and [17, 19]). Of the NK clones obtained from patient B, 52%expressed the CEACAM1 protein in low or high levels (FIG. 11), whereas69% of the NK cells derived from patient C expressed CEACAM1 in low orhigh levels (FIG. 11).

Activated bulk NK cultures were next assessed for the expression of CD3,CD16, CD56 and CEACAM1. The minor CEACAM1-positive population could notbe observed when the activated bulk NK cultures derived either from thehealthy sister or from an unrelated healthy donor (NK-Y) were analyzed(FIG. 12A). In contrast, an up-regulation of CEACAM1 was observed on thebulk activated NK cells derived from patient B. An up-regulation in theCEACAM1 expression was observed on bulk activated NK cells derived frompatient C that was associated with a moderate down-regulation in theexpression of the CD16 and a reduction in the CD56 expression (FIG.12A). The CD16 expression on the surface of NK cells obtained from thehealthy sister was lower than in the unrelated healthy donor;nevertheless, no CEACAM1 expression was observed (FIG. 12A).

CEACAM1-Mediated Inhibition of NK Cytotoxicity is Abrogated by SolubleCEACAM1

CEACAM1 controls NK autoreactivity in TAP2-deficient patients [20].Therefore, the maintenance of the CEACAM1-inhibitory interactions iscritical in these patients. CEACAM1-Ig was used to investigate theeffect of soluble CEACAM1 on NK-mediated killing. NK clones and bulkcultures were tested in killing assays against the 721.221 (.221) cellsand the CEACAM1-transfected .221 cells (.221/CEACAM1) as described [30].The bulk NK cultures obtained from patient B (NK-B) efficiently killedthe .221 cells (FIG. 12B), but were inhibited by the .221/CEACAM1 cells(FIG. 12B). This is in agreement with the unusually high expression ofthe CEACAM1 protein on the surface of NK cells derived from theTAP2-deficient patients (FIG. 11, 12A, and [20]). The inhibitionobserved with the bulk NK cells derived from the patients was the resultof the homophilic CEACAM1 interactions, as lysis was restored in thepresence of blocking anti-CEACAM antibodies (FIG. 12B). Remarkably, theCEACAM1-mediated inhibition was abrogated in the presence of the solubleCEACAM1-Ig in a dose-dependent manner, reaching maximal effect in 5?g/well (FIG. 12B). In addition, the presence of either CEACAM1-Ig orCD99-Ig did not affect the killing activity of CEACAM1− bulk NK culturesobtained from a healthy donor, thus ruling out nonspecific increasedkilling due to antibody-dependent cellular cytotoxicity (FIG. 12C).Similar results were obtained with CEACAM1+ NK cells pooled from otherhealthy donors (FIG. 12D).

TAP2-Deficient Patients have Decreased Level of Serum CEACAM1

The amount of the soluble CEACAM1 protein is normally around 300 ng/ml[22], but in pathologies such as obstructive jaundice it increases to1,500 ng/ml [22]. Induction of liver diseases in animal models leads toincreased soluble CEACAM1 protein in the serum [31]. Based on theblocking activity of CEACAM1-Ig (FIG. 12B, D), it is possible that serumCEACAM1 levels might be altered in TAP2-deficient patients. This issupported by other receptors that are also biologically active insoluble forms such as the Semaphorin CD100/Sema4D [32].

The levels of soluble CEACAM1 protein were tested in the patients' sera.CEACAM1 levels detected in the sera derived from unrelated healthydonors were similar to those previously described [22, 23] (FIG. 13A).In contrast, a striking decrease in the soluble CEACAM1 amount wasobserved in the sera derived from all three patients (FIG. 13A).Interestingly, although the amount of soluble CEACAM1 in the serumderived from the healthy mother was indeed significantly higher thanthat of the three patients, it was still significantly lower than normal(FIG. 13A). The TAP2 deficiency described here probably results from anautosomal recessive inherited defect, like other TAP2 deficiencies [24,33]. Hence, the patients are probably homozygous for the defect causingthe TAP2 deficiency and the mother is heterozygous. Therefore, the abovesignificant differences in serum CEACAM1 levels between unrelatedhealthy donors, the healthy mother and the patients might be linked tozygocity.

The bulk NK cultures were next tested against .221 cells and against the.221/CEACAM1 pre-incubated either with no serum, with patient-derivedserum or with serum derived from a healthy donor. The NK-B cells wereinhibited by the .221/CEACAM1 cells either in the presence of theautologous serum or when no serum was included in the assay (FIG. 13B).This inhibition was abrogated in the presence of serum derived from ahealthy donor (FIG. 13B), suggesting that the serum soluble CEACAM1 isable to block CEACAM1-mediated inhibition in vivo. No inhibition wasobserved when the CEACAM1-negative bulk NK cultures derived either froman unrelated healthy donor or from the healthy sister were used (FIG.13C, D). The presence of serum from the healthy donor caused a markedincrease in the killing activity of all NK cells tested (FIG. 13B-D).Similar activation of NK killing was observed with sera derived fromother healthy donors, whereas sera derived from patient C had no effect.Additional differences other than the presence of CEACAM1 exist mayexist.

Soluble CEACAM1 Protein is not Generated Via Membrane-BoundMetalloproteinasemediated Cleavage

The combination of increased expression of membrane-bound CEACAM1protein on cells derived from the TAP2-deficient patients [20], togetherwith the significantly lower amounts of soluble CEACAM1 in the sera ofthe patients (FIG. 13A), suggests that NK cells in the TAP2-deficientpatients have developed special mechanisms to inhibit NK activity. Thesoluble CEACAM1 protein found in the serum might be generated eitherfrom alternative splicing of CEACAM1 or from a cleavage of themembrane-bound CEACAM1 by membrane-bound metalloproteinase (MBMP).Whether MBMP is involved in the cleavage of CEACAM1 was tested. MBMPactivity is augmented in response to cell stimulation, e.g. by PMA [34,35], and inhibited in the presence of the inhibitor BB-94 [36]. Previousreports have demonstrated that MHC class I proteins are susceptible toexternal cleavage by MBMP [37]. Incubation of the NK-M or the NK-Y cellswith the PMA resulted in decreased MHC class I expression (FIG. 14A).This reduction in MHC class I expression was the result of MBMPactivity, because MHC class I expression level was restored in thepresence of the BB-94 inhibitor (FIG. 14A). As shown above andpreviously (FIGS. 10, 14B and [20]), the MHC class I protein expressionlevel on NK-B cells was significantly lower compared with the healthydonors. Surprisingly however, expression of MHC class I on the patientcells did not decrease in response to PMA (FIG. 14B). Similar resultswere obtained with bulk NK cultures derived from patients A and C. Thismay suggest that the specific MHC class I alleles expressed onTAP2-deficient cells are resistant to extracellular cleavage by MBMP.Alternatively, the activity or expression of the MBMP might be impairedin the TAP2− deficient cells.

To test the latter option, the NK-B, -M and -Y cells for the NKp46receptor with the 461-G1 mAb were stained [9, 20]. As reported [20],expression of NKp46 was detected on the normal NK cells, NK-M and -Y.However, the NK-B cells displayed a much weaker expression (FIG. 14C).Reduction in the NKp46 expression level was observed on NK cells derivedfrom both healthy donors and patients following incubation with PMA(FIG. 14C). The reduction in 461-G1 staining was induced by MBMPactivity as NKp46 expression was restored in the presence of the BB-94inhibitor (FIG. 14C). MBMP is active and functional in theTAP2-deficient patients.

Whether the surface CEACAM1 protein is regulated by MBMP-mediatedcleavage was also tested. The NK-Y bulk culture was obtained afterpooling more than 30 CEACAM1⁺ NK clones obtained from a healthy donor.Incubation of the various bulk NK cultures with PMA did not alter theCEACAM1 expression level (FIG. 14D). This indicated that themembrane-bound CEACAM1 protein expression level is not regulated by MBMPmediated cleavage.

Example 3 The Mechanisms Controlling NK Cell Autoreactivity inTAP2-Deficient Patients

The killing of natural killer (NK) cells is regulated by activating andinhibitory NK receptors that recognize mainly class I majorhistocompatibility complex (MHC) proteins. In transporter associatedwith antigen processing (TAP2)-deficient patients, killing of autologouscells by NK cells is therefore expected. However, none of theTAP2-deficient patients studied so far have suffered from immediateNK-mediated autoimmune manifestations. The present inventors havedemonstrated the existence of a novel class I MHC-independent inhibitorymechanism of NK cell cytotoxicity mediated by the homophiliccarcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)interactions. In this section the present inventors identify 3 newsiblings suffering from TAP2 deficiency. NK cells derived from thesepatients express unusually high levels of the various killer cellinhibitory receptors (KIRs) and the CEACAM1 protein. Importantly, thepatients' NK cells use the CEACAM1 protein to inhibit the killing oftumor and autologous cells. The inventors further show that the functionof the main NK lysis receptor, NKp46, is impaired in these patients. Inthis section the present inventors will show that the expression of theNKp46 receptor is severely impaired in a newly identified TAP2-deficientfamily and that the vast majority of activated NK cells derived fromthese patients use the CEACAM1 protein interactions to avoid tumor andautologous cell killing. These results indicate that NK cells inTAP2-deficient patients have developed unique mechanisms to reduce NKkilling activity and to compensate for the lack of class I MHC-mediatedinhibition. These mechanisms prevent the attack of self-cells by theautologous NK cells and explain why TAP2-deficient patients do notsuffer from autoimmune manifestations in early stages of life. (GalMarkel et al., The mechanisms controlling NK cell autoreactivity inTAP2-deficient patients, Blood, 1 Mar. 2004, Vol. 103, No. 5, pp.1770-1778. Pre-published online as a Blood First Edition Paper on Nov.6, 2003; DOI 10.1182/blood-2003-06-2114. This reference is herein nowincorporated by reference.)

Materials and Methods Patients

Patients A, B, and C are siblings (19, 14, and 9 years old,respectively). All patients share the same human leukocyte antigen (HLA)haplotype (A*03, B*07, Bw6, Cw*07, DRB1*15, DRB5, DQB1*06). Theypresented with severe diffuse bronchiectasis, sinusitis, and serousotitis media with no history of severe viral infections. TheInstitutional Review Board of Schneider Children's Medical Center ofIsrael approved these studies, and informed consent was providedaccording to the Declaration of Helsinki.

Generation of NK Clones and Phytohemagglutinin (PHA)-Induced T-CellBlasts

Isolation and culturing of NK clones and bulk NK cultures were performedas described.²² Isolation and culturing of T-cell populations wereperformed as described. 18

Antibodies and Immunoglobulin-Fused Proteins

The following monoclonal antibodies were used: monoclonal antibody (mAb)W6/32 and HP-1F7 directed against class I MHC molecules;anti-β₂-microglobulin mAb BBM-1, anti-CEACAM1 mAb 5F4,²³ anti-HLA-A3 GAPA3 mAb, anti-CD16 mAb B73.1.1, and anti-CD94 mAb HP-3D9 (Dako, Hamburg,Germany); the rabbit polyclonal anti-CEACAM1, CEACAM5, and CEACAM6antibodies (Dako) that block the CEACAM1 interactions^(15,18);anti-killer cell inhibitory receptor 2DL1 (anti-KIR2DL1) mAb EB6(ImmunoTech, Westbrook, Me.); anti-KIR2DL2 mAb GL183 (ImmunoTech);anti-leukocyte immunoglobulin-like receptor 1 (anti-LIR1) mAb HP-F1 (akind gift from Dr López-Botet; Immunologica, Barcelona, Spain);anti-HLA-DQ mAb G46-6 (Pharmingen, San Diego, Calif.); anti-HLA-DR mAbTU36 (Pharmingen); and the anti-NKG2D mAb (R&D Systems, Minneapolis,Minn.). The specificity of all anti-CEACAM antibodies was confirmedpreviously.¹⁸ The anti-NKp46 mAb 461-G1 (immunoglobulin G1 [IgG1]) wasgenerated by immunizing mice with the NKp46-Ig fusion protein. Thespecificity of this mAb was determined by fluorescence-activated cellsorter (FACS) analysis on NKp46 transfectants and on NK cells (freshlyisolated and IL-2 activated) (Table 4). The generation and production offusion proteins KIR2DL2-Ig, CD99-Ig, NKp46-Ig, NKp30-Ig, and NKp44-Igwere previously described.^(3,7,8)

Restoration of Class I MHC Expression by Transient B-Cell Line Fusion

Epstein-Barr virus (EBV)-transformed B-cell lines derived from thepatients were mixed either with the 721.174 (a TAP⁻ cell line),²⁴ withthe 721.45 (a TAP⁺ cell line with hemizygous MHC class I haplotype ofHLA-A2, HLA-B5, and HLA-Cw1),²⁵ or with EBV-transformed B-cell linesderived from other TAP1- or TAP2-deficient patients. Cells to be fusedwere mixed together at a 1:1 ratio at a final concentration of 10×10⁶/mLand incubated for 1 hour in RPMI containing 10% fetal calf serum (FCS)and 25 μg/mL PHA-P (Sigma, St Louis, Mo.). Cells were pelleted andresuspended in phosphate-buffered saline (PBS) containing 50%polyethylene glycol (PEG) 1500 (Sigma), and 5% dimethyl sulfoxide (DMSO)(Sigma). After incubation at 37° C. for 1 minute, cells were washed,resuspended in PBS, and incubated for a further 30 minutes at 37° C.After overnight incubation in RPMI containing 10% FCS, total fusionproducts were stained with GAP A3 monoclonal antibody. Cell mixtureswithout addition of PEG were used as negative control to rule outhumoral effect.

FACS Staining, Generation of F(AB′)₂ Fragments

FACS multistaining was performed as described.^(15,18) Conjugatedantibodies included Kat4c-fluorescein isothiocyanate (Kat4c-FITC) (IgG1;Dako), anti-CD56-phycoerythrin (PE) (IgG1; Dako), anti-CD3-CyChrome(IgG1; Pharmingen), anti-CD16-biotin (IgG1; Serotec, Raleigh, N.C.), andanti-NKG2D-PE (IgG1; R&D Systems). Controls were nonbindingisotype-matched fluorochrome-matched mAbs. Detection ofimmunoglobulin-fusion proteins was performed by means of thePE-conjugated secondary goat antihuman IgG antibodies with minimalcross-reaction (Jackson ImmunoResearch Laboratories, Bar Harbor, Me.),as described.^(15,18) All FACS data in all of the figures and tablespresented in this article were obtained with antibodies in the form ofF(ab′)₂. Digestion and purification of the F(ab′)₂ fragments wereperformed with the ImmunoPure F(ab′)₂ preparation kit (Pierce, Rockford,Ill.) according to the manufacturer's instructions.

Cytotoxicity Assays

The cytotoxic activity of NK cells against the various targets wasassayed in 5-hour ³⁵S-release assays, as described.²⁶ In experiments inwhich antibodies were included, the final mAb concentration was 20μg/mL. In all assays performed, the spontaneous release did not exceed25% of the maximal labeling.

Identification of a New Family of TAP2-Deficient Patients

A genetic defect in the class I MHC expression was identified in anArab-Israeli family. Patients A, B, and C (19-year-old female, 14- and9-year-old males, respectively) displayed clinical manifestationssimilar to those displayed by other TAP2-deficient patients.²⁷ The other5 sisters as well as the parents, who are first cousins, showed noclinical symptoms. NK clones were purified from all of the patients aswell from a healthy sister. Forty NK clones from each individual wereanalyzed by FACS for presence of class I MHC, class II MHC, andβ₂-microglobulin by use of the W6/32 mAb, the TU36 mAb, and the BBM-1mAb, respectively. A decrease in expression of class I MHC proteins andof β₂-microglobulin (approximately 20-fold) was observed on the surfaceof NK clones derived from the patients as compared with those derivedfrom the healthy sister (Table 2). Analysis of class II MHC proteinexpression revealed only 3-fold (patient A) or 2-fold (patients B and C)reduction compared with the healthy sister (Table 2). Similar resultswere obtained when different types of cells, such as T cells andmonocytes, were used.

To identify the genetic defect responsible for the impaired expressionof class I MHC proteins observed in these patients, an EBV-transformedB-cell line was made from each patient (EBV-A, EBV-B, and EBV-C) andfrom their healthy mother (EBV-mother). The EBV-transformed B-cell lineswere further analyzed for the expression of class I MHC, class II MHC,β₂-microglobulin, and HLA-A3 by using the W6/32 mAb, the TU36 and G46-6mAbs, BBM-1 mAb, and the GAP A3 mAb, respectively. In agreement with theresults described in the preceding paragraph, a dramatic down-regulation(approximately 20-fold) of class I MHC proteins and of β₂-microglobulinsurface expression was observed on EBV-A, EBV-B, and EBV-C as comparedwith EBV-mother (Table 3). There was no significant difference in theexpression of class II MHC proteins, such as HLA-DQ and HLA-DR (Table3). Notably, the HLA-A3 protein was completely absent from the surfaceof EBV-A, EBV-B, and EBV-C, but not from EBV-mother (all patients andtheir mother express HLA-A3; see “methods this section”). There is aslight expression of class I MHC detected by W6/32, indicating thatclass I MHC proteins other than HLA-A3 are still expressed in low levelson the patient EBV cells (Table 3).

Since HLA-A3 was not detected on the patients' EBV cells, an assay thatis based on the restoration of HLA-A3 expression following correction ofthe class I MHC biosynthetic pathway via PEG-mediated fusion of EBV-A,EBV-B, or EBV-C with various cell lines was next employed. The 721.45cells that were used in some of the fusion experiments were hemizygousfor class I MHC expression (HLA-A2, HLA-B5, and HLA-Cw1). Therefore,specific monitoring was required to discriminate between the class I MHCreconstitution (monitored by GAP A3) and the endogenous expression ofthe class I MHC proteins on 721.45 cells.

Cells were fused, and the total cell mixture was analyzed with the useof the GAP A3 mAb. As not all mixed cells were fused, the fused cellswere identified by the presence of HLA-A3 protein. Fusion of EBV-A,EBV-B, or EBV-C either with the 721.45 cell line that expresses bothTAP1 and TAP2 subunits or with cells deficient in TAP1, resulted in theemergence of an HLA-A3⁺ population (FIG. 15). In contrast, fusion ofEBV-A, EBV-B, or EBV-C with 721.174 cell line (0.174), deficient forTAP1 and TAP2 or with B cells derived from other TAP2-deficientpatients, failed to restore HLA-A3 expression (FIG. 15). The fact thatmixture of EBV-A, EBV-B, or EBV-EBV-C with the various cell lineswithout addition of PEG failed to reconstitute HLA-A3 expression and inaddition to the fact that some of the fusions performed did not restoreHLA-A3 expression (FIG. 15) rule out the possibility of humoral effect.The genetic defect is in the TAP2 protein.

Impaired Expression and Function of NKp46

NKp46 is considered to be the main NK killing receptor for NK cells andis uniquely expressed on all NK cells.²³ Expression of NKp46 on freshlyisolated NK cells was monitored by using an anti-NKp46 mAb (461-G1).Relatively low levels of NKp46 were observed on the bulk NK cellsisolated from the healthy sister (MFI=24) (FIG. 16A). At this stageblood samples could no longer be obtained from patient C owing to theseverity of his clinical conditions. The NKp46 receptor could hardly bedetected on the surface of more than 85% of the NK cells isolated frompatient A (MFI=7) and on more than 60% of the NK cells isolated frompatient B (MFI=10) (FIG. 16A). The function of the NKp46 receptor wasassayed concomitantly against 721.221 cells, in which the lysis iscontrolled by the NKp46 receptor.⁵ In accordance with the stainingresults (FIG. 16A), very low killing was observed with bulk freshlyisolated NK cells derived from patient A; relatively moderate killingwas observed with NK cells from patient B; and relatively efficientkilling was observed with NK cells from the healthy sister (FIG. 16B).

IL-2-activated NK clones from the patients and from the healthy sisterwere next generated. No major difference in the NKp46 expression wasobserved between freshly isolated and IL-2-activated NK cells (FIG. 16A;Table 4). Activated NK clones were analyzed for NKp46 expression and forcytotoxicity against .221 target cells. A reduction in the NKp46expression was observed in NK clones derived from all of the patients ascompared with the healthy sister. All 30 NK clones (100%) derived frompatient A did not express the NKp46 protein; 19 (39%) of 49 clones and56 (80%) of 70 clones derived from patient B and patient C,respectively, were also NKp46⁻ (Table 4). The absence of the NKp46receptor on these NK clones was correlated with poor cytolytic activityagainst .221 cells (Table 4). NKp46⁻ clones were not observed in thehealthy sister (Table 4). On the other hand, 29 (61%) of 49 NK clonesderived from patient B, 14 (20%) of 70 from patient C, and 30 (100%) of30 from the healthy sister were NKp46⁺ (Table 4). The level of the NKp46expression was similar in all positive clones (Table 4). Accordingly,the NKp46⁺ NK clones displayed efficient cytotoxic activity against .221target cells (Table 4). Efficient killing of other cells types such asEBV-A, EBV-B, EBV-C, 293T, or RPMI 8866 was also observed when assayedagainst the NKp46⁺ clones.

Activated NK Clones Derived from the TAP2-Deficient Patients ExpressUnusually High Levels of CEACAM1- and Class I MHC-Recognizing Receptors

The killing of targets by NK cells derived from the TAP2-deficientpatients can be reduced by the diminished NKp46 expression. However, 61%and 20% of the clones in patients B and C, respectively, still expressedNKp46 (Table 4). The present inventors hypothesized the existence of aclass I MHC-independent inhibitory mechanism in their patients thatcontrols NK autoreactivity and examined whether CEACAM1 interactionswere involved in controlling the killing activity of activated NK cellsin TAP2-deficient patients.

Peripheral blood lymphocytes (PBLs) were isolated from all 3 patientsand the healthy sister and analyzed by multistaining for the expressionof the CD3, CD16, CD56, and CEACAM1. All patients had normal values oflymphocytes in their peripheral blood and a normal lymphocytedistribution, including T, NK, and NKT cells. Thus, the low levels ofclass I MHC proteins (Tables 2, 3) were probably sufficient to selectfor the development of normal numbers of lymphocytes. PBLs from all 4donors were also tested for the expression of the CEACAM1 protein.Little or no expression of the CEACAM1 protein was observed among allfresh PBLs.

Activated NK clones were generated from the 3 patients and from thehealthy sister. Sixty NK clones from each individual were assessed forCEACAM1 expression by using the 5F4 mAb. The NK clones from eachindividual were sub grouped according to the CEACAM1 expression level(negative, low, or high). Low levels of CEACAM1 expression (MFI around8) had already been observed on NK clones and proved sufficient toconfer protection.^(15,16) The high expression level of CEACAM1 (MFIaround 30) observed on the surface of NK clones had not been observedbefore. 53 (88%) of 60 NK clones obtained from the healthy sister werenegative for CEACAM1 (Table 5). In contrast, virtually all of the NKclones (98%) obtained from patient A expressed the CEACAM1 protein inunusually high levels. Of the 60 NK clones obtained from patient B, 43(71%) expressed the CEACAM1 protein in low or high levels (43% and 28%,respectively; Table 5) whereas 44 (73%) of 60 NK cells derived frompatient C expressed CEACAM1 in low or high levels (23% and 50%,respectively; Table 5).

In addition, all of the NK clones were stained for the presence of CD16,KIR2DL1, KIR2DL2, CD94, or LIR1. In each individual, the total NK cloneswere further sub classified in each CEACAM1 subgroup according to thestaining intensity of each receptor (negative, dim, and bright), and themean MFI±SD was calculated accordingly. The overall percentages of NKclones from each individual expressing KIR2DL1, KIR2DL2, and LIR1 wascompared. KIR2DL1 was expressed on 62%, 77%, and 72% of the NK clonesobtained from patients A, B, and C, respectively, compared with only 25%of the NK clones from the healthy sister (Table 5). KIR2DL2 wasexpressed on 65%, 65%, and 85% of the NK clones obtained from patientsA, B, and C, respectively, compared with only 35% of the NK clones fromthe healthy sister (Table 5). Finally, the LIR1 was expressed on 67%,68%, and 60% of the NK clones obtained from patients A, B, and C,respectively, compared with only 15% of the NK clones from the healthysister (Table 5). Expression of all inhibitory NK receptors tested wasup-regulated on the NK cells derived from the patients. The increase inthe percentage of NK clones derived from the patients that express classI MHC-recognizing inhibitory receptors is statistically significant whencompared with the healthy sister: KIR2DL1 (P=0.01), KIR2DL2 (P=0.04),and LIR1 (P=0.02). Further analysis reveals that the receptor expressionlevel is also increased among NK clones obtained from the patients ascompared with those obtained from the healthy sister. Bright expressionof KIR2DL1 was observed on 24 of 60, 32 of 60, and 33 of 60 NK clonesobtained from patients A, B, and C, respectively, but not on any of the60 NK clones obtained from the healthy sister (P=0.003) (Table 5).Similarly, bright expression of KIR2DL2 was observed on 21 of 60, 34 of60, and 37 of 60 NK clones obtained from patients A, B, and C,respectively, as opposed to only 16 of 60 obtained from the healthysister (P=0.06) (Table 5). Patients of in the present section expressthe Cw7 protein, which is recognized by KIR2DL2.³⁰ A statisticallysignificant bright expression of KIR2DL1 but not KIR2DL2 was observed inthe patients' NK clones, suggesting that the expression level of thevarious NK receptors is somehow shaped by the appropriate MHC proteins(Table 5). Thus, the low levels of class I MHC proteins in the patientshave resulted in an impaired repertoire of inhibitory receptors,manifested not only in the increased percentages of positive clones butalso in the higher expression levels.

Expression of the CD94 receptor was observed on 95%, 97%, and 92% of theNK clones obtained from patients A, B, and C, respectively, and on 100%of the NK clones from the healthy sister (P=0.66) (Table 5). Theexpression of CEACAM1 is confined mainly to the CD16⁻ subset of NKcells.^(15,18) Expression of CD16 was observed on 83%, 98%, and 87% ofthe NK clones obtained from patients A, B, and C, respectively, and on90% of the NK clones from the healthy sister (P=0.29) (Table 5). CEACAM1expression on NK clones derived from the healthy sister was restrictedmainly to the CD16⁻ cells (6 of 7 CEACAM1^(dim) NK clones; Table 5). Incontrast, 50 of 59, 42 of 43, and 37 of 44 NK clones derived frompatients A, B, and C, respectively, expressed both CD16 and CEACAM1(P=0.007) (Table 5). There was observed expression of CEACAM1 on bothCD16⁻ and CD16⁺ NK clones derived from the patients (Table 5).

3.5 CEACAM1 interactions protect autologous PHA-induced T-cell blastsfrom NK cell-mediated killing

The functional significance of CEACAM1 expression was assayed withclones capable of killing .221 cells (Table 4). As the CEACAM1 proteinbinds via homophilic interactions to other CEACAM1proteins,^(15,18,31,32) the various NK clones were tested for killingagainst .221 cells expressing the CEACAM1 protein.^(15,18) Inhibition ofkilling was observed when CEACAM1⁺ NK clones were used (representativeclone in FIG. 17A). This inhibition was the result of CEACAM1interactions, as lysis was restored when anti-CEACAM F(ab′)₂ antibodieswere included in the assay (FIG. 17). No inhibition was observed whenCEACAM1⁻ NK clones were used (FIG. 17B).

It was nest determined whether normal, nonvirally infected, PHA-inducedT-cell blasts will be killed by the patients' NK cells. In agreementwith reports demonstrating that activated T cells express the CEACAM1protein,^(23, 29) expression of CEACAM1 was observed on all PHA-inducedT-cell blasts derived from all patients (FIG. 18A). The expression levelof the CEACAM1 protein on the PHA-induced T-cell blasts derived from theTAP2-deficient patients was approximately 5-fold higher as compared withthe PHA-induced T-cell blasts obtained from the healthy sister (FIG.18A). Low levels of class I MHC protein expression were observed onPHA-induced T-cell blasts derived from patients compared with thehealthy sister (FIG. 18A). Staining of the various PHA-induced T-cellblasts for the presence of ligands for the NKp46, NKp44, and NKp30receptors with the use of immunoglobulin-fusion proteins was negative(FIG. 18B). NKp46-Ig, NKp30-Ig, and NKp44-Ig did, however, recognizetumor targets such as LnCap (FIG. 18B) or other cell lines.^(7,8) Thecellular ligands for these receptors may be either not expressed or maybe expressed in low levels on the surface of the PHA-induced T-cellblasts. The expression of other lysis ligands such as MICA is present onthe PHA-induced T-cell blasts.³³

CEACAM1⁺ NK clones from each patient were assayed for lysis against thevarious PHA-induced T-cell blasts. All CEACAM1⁻ NK clones and bulkcultures derived from the healthy sister killed the TAP2-deficientPHA-induced T-cell blasts (see, e.g., “NK Sister CEACAM1⁻” in FIG. 18C).Similar results were obtained with bulk NK cells and clones obtainedfrom other healthy donor. None of the tested NK clones or bulk culturesderived from the patients killed their autologous PHA-induced T-cellblasts (see, e.g., NK A, NK B, and NK C in FIG. 18C). Blockingantibodies were included in the assay to test whether the lack ofself-killing is because of CEACAM1- or class I MHC-mediated inhibition.The PHA-induced T-cell blasts were preincubated with or without theanti-CEACAM antibodies, the control antiubiquitin antibodies, HP-1F7mAb, or the control 12E7 mAb. A significant enhancement of the killingactivity of the patients' NK clones (A, B, and C) was observed when theF(ab′)₂ fragments of anti-CEACAM antibodies were included in the assay,either alone or in combination with the anti-class I MHC mAb HP-1F7(FIG. 18C). Similar results were obtained regardless of whether the NKclones tested expressed the NKp46 receptor. No effect was observed whenthe F(ab′)₂ fragments of the anti-class I MHC mAb HP-1F7 were included(FIG. 18C). The low expression level of class I MHC proteins was notenough to confer protection. Killing was not restored when the controlF(ab′)₂ fragments of either the polyclonal antiubiquitin antibodies orthe 12E7 mAb were used (FIG. 18C). These results indicate thatself-attack of the autologous PHA-induced T-cell blasts by NK clonesderived from the patients is prevented by the homophilic CEACAM1inhibitory interactions.

Autologous NK clones negative for CEACAM1 expression were unable to killthe autologous PHA-induced T-cell blasts (see, e.g., patient C'sCEACAM1⁻ NK cells, which express NKp46; FIG. 18C). This property isunique to NK cells derived from the patients, as CEACAM1⁻ NK cells fromthe healthy sibling efficiently attacked self-cells following MHC classI blocking (FIG. 18C-D).

The NK clones and bulk cultures obtained from patients A, B, and C andthe healthy sister were next tested in killing assays againstPHA-induced T-cell blasts derived from the healthy sister. The variouscells were treated as described in the text discussion of FIG. 18C.CEACAM1⁻ NK clones derived from the healthy sister were unable to killthe autologous PHA-induced T-cell blasts. The inhibition was the resultof class I MHC interactions, as the F(ab′)₂ fragments of HP-1F7 mAbincluded in the assay, either alone or in combination with the F(ab′)₂fragments of anti-CEACAM antibodies, abolished this inhibition (FIG.18D). When CEACAM1⁺ NK clones derived from the TAP2-deficient patientswere used, lysis of the sister's PHA-induced T-cell blasts could becompletely restored only when the F(ab′)₂ fragments of both the HP-1F7and anti-CEACAM antibodies were used (FIG. 18D). Partial restoration ofkilling was observed when either the CEACAM1 or the class I MHCinteractions were disrupted, indicating that both inhibitory mechanismsprevent the killing of normal PHA-induced T-cell blasts. Similar resultswere obtained when CEACAM1⁺ NK clones derived from the healthy sisterwere used.

CEACAM1 protein is up-regulated on NK cells derived from some melanomapatients and from decidua.^(15,18) The vast majority of activated NKcells derived from TAP2-deficient patients express the CEACAM1 proteinin high levels, the expression of the CEACAM1 protein is restricted topatient-derived NK cells with the ability to kill self-cells, and theCEACAM1 protein is capable of inhibiting NK killing. NK cells derivedfrom TAP2-deficient patients have developed or acquired a uniquemechanism to control the killing of self-cells by using the CEACAM1interactions.

TABLE 2 Expression of MHC proteins on NK clones Antibody Patient APatient B Patient C Healthy sister Secondary F(ab′)₂  3.2 ± 0.7 2.2 ±0.3  2.8 ± 0.4  3.4 ± 0.8 background W6/32 F(ab′)₂ 36.7 ± 8.2  23 ± 6.530.7 ± 8.0  629 ± 183 BBM-1 F(ab′)₂  7.7 ± 2.5 5.3 ± 1.7  7.1 ± 1.6 130± 30 MHC class II 33.1 ± 3.6 54.3 ± 9.6  54.6 ± 7.7 94.7 ± 9.1 F(ab′)₂

Forty NK clones were isolated from each of the 3 patients and from thehealthy sister. Clones were stained by various mAbs as indicated andanalyzed by FACS. All antibodies used were in the form of F(ab′)₂fragments. Background was the secondary mAb in the form of F(ab′)₂. Dataare presented as the median fluorescence intensity (MFI) of 40clones±standard deviation (SD). All 160 clones were stained at the sametime.

TABLE 3 Expression of MHC proteins on EBV-transformed B-cell lines CellsBackground W6/32 BBM-1 HLA-DQ HLA-DR HLA-A3 EBV-A 2.5 ± 0.5   122.5 ±4.9 12.5 ± 2.1 22.5 ± 2.1 91.5 ± 23.3 2.5 ± 0.5 EBV-B 3 ± 0.5 106.5 ±2.1   8 ± 4.2   24 ± 2.8 99.5 ± 14.8 2.8 ± 0.6 EBV-C 2 ± 0.4   76 ± 1.47.35 ± 2.9 29.5 ± 4.9 114.5 ± 17.7  3.0 ± 0.5EBV-transformed B-cell lines were made from the indicated individuals.Cell lines were stained by various mAbs in the form of F(ab′)₂ asindicated and analyzed by FACS. Background was the secondary mAb in theform of F(ab′)₂. Data are presented as the average MFI of 3 independentexperiments±SD.

TABLE 4 Impaired expression and function of NKp46 on activated NK clonesPatient A Patient B Patient C Healthy sister .221 .221 .221 .221 NKp46,cells NKp46, cells NKp46, cells NKp46, cells MFI killed, % MFI killed, %MFI killed, % MFI killed, % NKp46⁻ 2.2 ± 0.9 6.7 ± 2.9  1.7 ± 0.6 7.2 ±3.7 1.8 ± 0.7 4.8 ± 2.1 N/O N/O (30/30) (30/30) (19/49) (19/49) (56/70)(56/70) NKp46⁺ N/O N/O 16.5 ± 6.3  25 ± 5.9 13.5 ± 6   20.9 ± 6   16.9 ±5 26.5 ± 5.3 (29/49) (29/49) (14/70) (14/70) (40/40) (40/40)

Activated NK clones were prepared from each individual as indicated.Clones were stained for NKp46 expression by means of the 461-G1 mAb inthe form of F(ab′)₂ and concomitantly tested for cytotoxic activityagainst .221 cells. NKp46 expression is presented as the mean MFI ofdifferent NK clones±SD. Cytotoxic activity is presented as the meanpercentage of .221 cells killed by NK clones. The data represent meanpercentage of cells killed±SD. The number of NK clones included in theanalysis out of total NK clones tested in each group are indicated inparentheses.

N/O indicates not observed.

Example 4 CD66A Interactions Between Human Melanoma and NK Cells: ANovel Class I MHC-Independent Inhibitory Mechanism of Cytotoxicity

NK cells are able to kill virus-infected and tumor cells via a panel oflysis receptors. Cells expressing class I MHC proteins are protectedfrom lysis primarily due to the interactions of several families of NKreceptors with both classical and nonclassical class I MHC proteins. Thepresent inventors show that a class I MHC-deficient melanoma cell line(1106mel) is stained with several Ig-fused lysis receptors, suggestingthe expression of the appropriate lysis ligands. This melanoma line wasnot killed by CD16-negative NK clones. The lack of killing is shown tobe the result of homotypic CD66a interactions between the melanoma lineand the NK cells. Furthermore, 721.221 cells expressing the CD66aprotein were protected from lysis by YTS cells and by NK cellsexpressing the CD66a protein. Redirected lysis experiments demonstratedthat the strength of the inhibitory effect is correlated with the levelsof CD66a expression. Finally, the expression of CD66a protein wasobserved on NK cells derived from patients with malignant melanoma.These findings suggest the existence of a novel class I MHC-independentinhibitory mechanism of human NK cell cytotoxicity. This may be amechanism that is used by some of the class I MHC-negative melanomacells to evade attack by CD66a-positive NK cells. (Gal Markel et al.,CD66a Interactions Between Human Melanoma and NK Cells: A Novel Class IMHC-Independent Inhibitory Mechanism of Cytotoxicity, The Journal ofImmunology, 2002, 168: 2803-2810. This reference is herein incorporatedby reference.)

Materials and Methods Cells and MAB

The cell lines used in this section are the class I MHC-negative humancell line 721.221, the YTS NK tumor line, and various MHC classI-negative and -positive human melanoma cell lines. NK cells wereisolated from PBL using the human NK cell isolation kit and the autoMACSinstrument (Miltenyi Biotec, Auburn, Calif.). For the enrichment ofCD66a-positive NK cells, isolated NK cells were further purified bydepletion of CD16-positive NK cells using anti-CD16 mAb 3G8 and theautoMACS instrument. NK cells were grown in culture as described. Theproduction and specificity of anti-NKp44 and NKp46 sera were asdescribed. The mAbs used in this section were mAb W632, directed againstclass I MHC molecules, the mAb anti-CD66 a,b,c,e Kat4c (purchased fromDAKO, Carpenteria, Calif.), anti-CD66a mAb 5F4, and the rabbitpolyclonal anti-CD66a,c,e Abs (purchased from DAKO). The anti-CD99 mAb12E7, used as a control, was from the Hopital de L'Archet, Nice, France.The anti-CD56 mAb (BD PharMingen, San Diego, Calif.) was also used ascontrol.

Cytotoxicity Assay and Ig Fusion Proteins

The cytotoxic activity of YTS and NK cells against the various targetswas assayed in 5-h ³⁵S release assays as described. In experiments inwhich mAb were included, the final mAb concentration was 10 μg/ml, or 40μl/ml in cases where rabbit polyclonal Abs were used. Redirected lysisexperiments were performed as described. The production of CD99-Ig,CD16-Ig, NKp30-Ig, NKp44-Ig, and NKp46-Ig fusion proteins by COS-7 cellsand purification on a protein G column were as described.

Quadruple Staining

For quadruple staining, the following fluorochrome-conjugated mAbs wereused: FITC-conjugated anti-CD66 Kat4c mAb (DAKO), PE-conjugatedanti-CD56 mAb (BD PharMingen), and CyChrome-conjugated anti-CD3 (BDPharMingen). As the fourth color, biotinylated anti-CD16 mAb (Serotec,Oxford, U.K.) was used, followed by streptavidin-Cy5 (JacksonImmunoResearch Laboratories, West Grove, Pa.) as a second reagent. Toblock nonspecific binding, cells were first incubated for 1 h on icewith 25% human serum and then incubated with the various Abs.

Generation of YTS and 721.221 Cells Expressing CD66A

The primers used for the amplification of CD66a cDNA needed for thetransfection of 721.221 cells were as follows: 5′ primer,CCCAAGCTTGGGGCCGCCACCATGGGGCACCTCTCAGCC (including the HindIIIrestriction site), and 3′ primer, GGAATTCCTTACTGCTTTTTTACTTCTGAATA(including the EcoRI restriction site). cDNA was cloned into the pCDNA3vector (Invitrogen, San Diego, Calif.) and transfected into 721.221cells as described. For transfection into YTS cells, CD66a cDNA wasamplified by PCR using the 5′ primer GGAATTCCGCCGCCACCATGGGGCACCTCTCAGCC(including the EcoRI restriction site) and the 3′ primerGCGTCGACTTACTGCTTTTTTACTTCTGAATA (including the SalI restriction site).For amplification of the CD66aTrunc cDNA, the same 5′ primer was used,with the 3′ primer GCGTCGACATCTTGTTAGGTGGGTCATT. Amplified fragmentswere cloned into the pBABE retroviral vector and transfected into YTScells as described.

Expression of Various Lysis Ligands, CD66A, and Class I MHC Proteins onHuman Melanoma Cells

The roles of NKp30, NKp44, NKp46, and CD16 receptors in NK recognitionof various melanoma cells deficient in class I MHC expression (exceptfrom LB33melA1, used as a control) were studied by production of fusionproteins in which the extracellular domains of NKp30 NKp44, NKp46, andCD16 were fused to the Fc portion of Ig. cDNA encoding the extracellulardomains of CD99 fused to the human IgG1 DNA was used as a control. TheIg fusion proteins were incubated with the various melanoma cells andanalyzed for binding by indirect immunostaining as previously described(15). In general, the highest staining of the melanoma cells wasobserved with the NKp30-Ig and NKp44-Ig fusion proteins (Table 6).Little staining of all Ig fusion proteins was observed with LB33melA1cells, a cell line that is hardly killed by NK cells. All other celllines that can be killed by NK cells were stained to various degreeswith the Ig fusion proteins (Table 6).

IL-2-activated CD16-negative NK cells inefficiently kill 1106mel cells.Surprisingly, 1106mel cells probably express the ligands for all the NKlysis receptors tested, including CD16, NKp30, NKp44, and NKp46 (Table6). Thus, killing of 1106mel cells was expected to occur even whenCD16-negative NK cells, which express the NKp44 and NKp46 receptors,were used. The present inventors hypothesized the existence of a classI-independent mechanism of inhibition of NK cell cytotoxicity thatcontrols the lysis of 1106mel cells. The present inventors hypothesizedthat this inhibitory mechanism should include a protein that isexpressed mainly on the surface of IL-2-activated CD16-negative NK celland is expected to deliver an inhibitory signal via the ITIM. The CD66a(carcinoembryonic Ag CAM1) protein is expressed primarily onCD16-negative NK cells, it contains ITIM sequences, and it can bind in ahomotypic/heterotypic manner to various CD66 proteins. The inhibitoryeffect of the CD66a protein on human NK cell cytotoxicity wasinvestigated by the present inventors.

The present inventors tested whether the expression of CD66a moleculecan be detected on the surface of various melanoma cell lines and NKcells. Remarkably, all seven class I MHC-negative melanoma cell linestested expressed the CD66a protein at moderate or high levels (Table 7).No expression of MHC class I protein (detected with the W632 mAb) wasobserved among these cell lines, except from the LB33melB1 cell linethat expresses the HLA-A24 protein only. In contrast, 40% of the class IMHC-positive melanoma lines tested showed little or no staining for theCD66a protein (Table 7).

Recognition of CD66A Expressed on 1106mel by CD66A on CD16-Negative NKCells Leads to Inhibition of Lysis

The CD66a isoform is expressed on CD16-negative NK cells. Whether1106mel cells would be protected from lysis by CD16-negative NK cellsexpressing the CD66a protein was tested. For the generation ofCD16-negative NK cells expressing CD66a, NK cells were first isolatedfrom PBL of various healthy donors and then depleted from CD16-positiveNK cells by using the anti-CD16 mAb 3G8 (as described in Materials andMethods of this section). Of 63 CD16-negative clones tested, 28 (45%)expressed the CD66a protein. In rare cases (2%) CD66a expression couldbe detected on the surface of CD16-positive NK clones (see FIG. 23). Thepercentage of CD16-negative, CD66a-positive NK cells can vary amongdifferent donors after activation. Various NK clones were then tested inkilling assays against 1106mel cells. Efficient killing of 1106mel cellswas observed with CD16⁺CD66a⁻ NK clones (FIG. 19, A and B). The additionof anti-CD66 polyclonal Abs or the control 12E7 mAb (incubated witheither effector or target cells) had little or no effect (FIG. 19↓, Aand B). Similar results were obtained when CD16⁻CD66a⁻ NK clones wereused (see FIG. 23↓). In agreement with other observations, littlekilling of 1106mel cells was observed when CD16⁻CD66a⁺ NK cells (forexample, clone 163) were used (FIG. 19↓, C and D), whereas effectivekilling (32.4%) of the CD66-deficient NK-sensitive 721.221 cell line wasobserved. The low rate of 1106mel killing observed was the result of theinhibition mediated by the CD66a homotypic interactions, as lysis of1106mel cells was restored when either the effector or the target cellswere incubated with anti-CD66a polyclonal Abs (FIGS. 19, C and D). Theanti-CD66 polyclonal Abs specifically stained all cells that werepositive for CD66a expression (NK clones, melanomas, and varioustransfectants) and did not stain cells that were negative for CD66aexpression (for example, CD66a-negative NK clone). The controls, 12E7mAb or polyclonal Abs from rabbit immunized with purified ubiquitin, hadlittle or no effect (FIG. 19). Similar results were obtained whenCD16⁺CD66a⁺ NK clones were used (see FIG. 23). Reversal of theCD66a-mediated inhibition was also observed even when the LB33melB1 cellline was used as a target. This cell line expressed the lowest levels ofCD66a proteins among all seven class I-negative melanoma cells tested(Table 7). No inhibition of lysis by CD66a-positive NK cells wasobserved when these clones were incubated with the 1259mel melanomaline, a cell line that is efficiently killed by CD16-negative NK cells(FIG. 19E). The expression levels of the lysis ligands for CD16, NKp30,NKp44, and NKp46, detected by the Ig fusion proteins were similar tothose of 1106mel cells. One possible explanation is that other lysisligands for other lysis receptors exist on the surface of 1259mel cells,and the combined effect of all lysis receptors overcomes theCD66a-mediated inhibition.

The 721.221 Cells Expressing the CD66A Protein are Protected from Lysisby CD66A-Positive NK and YTS Cells

To directly test the role of the CD66a protein in inhibition of NK cellcytotoxicity, 721.221 target cells and YTS effector cells (bothdeficient for CD66a expression; FIG. 20.) were transfected with theCD66a cDNA. Several clones of 721.221 cells expressing various levels ofCD66a protein (.221/CD66a) were obtained. Two representative clones,expressing either low (.221/CD66a^(low)) or high (.221/CD66a^(high))levels, are shown in FIG. 20. YTS cells expressing either CD66a(YTS/CD66a) or CD66a in which the cytoplasmic tail of the molecule wastruncated not to include the ITIMs (YTS/CD66aTrunc) were also generated(FIG. 20). The expression level of the CD66a protein on YTS cells wassimilar to the physiological level of expression on an average primaryNK clone (median fluorescence intensity (MFI) was >2-fold over thebackground; a representative clone is shown in FIG. 20). YTStransfectants expressing higher levels of CD66a protein could not beobtained. Transfectants were next tested in cytotoxicity assays.Inhibition of YTS/CD66a killing was observed when cells were incubatedwith .221/CD66a^(high) in all E:T cell ratios tested (FIG. 21). Thepercentages of killing of other targets, including the parental 721.221or the .221/CD66a^(low) may be considered similar. Similar results wereobtained with other YTS and 721.221 cells expressing similar levels ofCD66a.

Lysis of all target cells tested against YTS cells transfected with thepBABE vector alone (YTS/MOCK) was similar. The CD66a inhibitory signalis probably transduced via the ITIM sequences, as no inhibition of lysisby YTS/CD66aTrunc was observed, even when these cells were incubatedwith 721.221/CD66a^(high) cells (FIG. 21). The low level of CD66aexpression on target cells (.221/CD66a^(low)) did not confer protection(FIG. 21). The inhibition of lysis of .221/CD66a^(high) cells byYTS/CD66a was the result of the CD66a interactions, as lysis wasrestored when anti-CD66 Abs were included in the assays (incubatedeither with the effector cells (FIG. 22A) or with the target cells. Thecontrols, 12E7 mAb or rabbit polyclonal Abs directed against ubiquitin,had no effect.

Lysis experiments were also performed with NK clones positive ornegative for the expression of CD66a. NK clones were prepared asdescribed above and tested against .221/CD66a^(high) cells.CD66a-dependent inhibition of lysis of .221/CD66a^(high) cells wasobserved when CD66a-positive NK cells were used (a representative cloneis shown in FIG. 22B). No inhibition of lysis of .221/CD66a^(high) cellswas observed when CD66a-negative NK clones were used (representativeclone is shown in FIG. 22C).

Levels of CD66A Expression on Both Effector and Target Cells areImportant for Effective Inhibition

One potential explanation for the moderate inhibition observed when.221/CD66a^(high) cells were incubated either with YTS/CD66a or withCD66a⁺ NK cells is the level of CD66a expression on both target andeffector cells. Indeed, no inhibition of lysis was observed when.221/CD66a^(low) cells were used (FIG. 21), moderate inhibition wasobserved when .221/CD66a^(high) cells were used (FIG. 21), and stronginhibition of lysis was observed when 1106mel cells were used (FIGS. 19,C and D). The 1106mel cell line expresses the CD66a protein at a level10-fold higher than that of the .221/CD66a^(high) transfectants (Table 7and FIG. 20). 721.221 cells expressing the CD66a protein at a higherlevel than the transfectant presented in FIG. 20 could not be obtained.Thus, the level of CD66a expression on target cells is important foreffective inhibition of both YTS and NK cells.

To correlate the level of expression of CD66a on NK cells and thestrength of inhibition, various NK clones (positive or negative forCD16) expressing different levels of CD66a were used. The redirectedlysis of P815 cells was induced with either anti-CD16 mAb or anti-NKp44and -NKp46 sera depending whether the NK clone tested expressed the CD16protein. A direct correlation was observed between the level of CD66expression on the surface of the NK clones and the percentage ofinhibition of redirected lysis (FIG. 23). The level of CD66a expressionhad to be at least 2-fold above the background staining for efficientinhibition to occur (FIG. 23).

Elevation of CD66A Expression on NK Cells Derived from Melanoma Patients

The in vivo significance of the CD66a interactions was studied by thestaining of NK cells derived from either metastasized lymph nodes orperipheral blood. The lymph node of patient M-169 was infiltrated withmelanoma cells, highly positive for the CD66a expression (MFI, 247). Thelymph node was surgically removed, and lymphocytes in direct contactwith the tumor cells were obtained after digestion and density gradientseparation. Quadruple staining of the lymphocytes was performed for theexpression of the CD16, CD3, CD56, and CD66 receptors. Remarkably,12.85% of the NK cells (CD56⁺CD3⁻) obtained from M-169 lymph nodeexpressed the CD66a protein (FIG. 24A). A total of 10.5% of the NK cellsobtained were CD16⁻CD66⁺, and 2.35% were CD16⁺CD66⁺. The MFI of theCD66a-positive NK population was 8-fold above background, which issufficient for effective inhibition (an MFI>2-fold above background isneeded; see FIG. 23). Similar results were obtained when peripheralblood NK cells derived from patient 3 were analyzed with the samequadruple staining; 14.8% of the NK cells were CD66a positive, and theMFI of this NK population was 7.5-fold above background (FIG. 24B).

In contrast, little or no CD66a expression was observed among NK cellsderived from the metastasized lymph node of patient M-172 (FIG. 24C).Strikingly, the infiltrating M-172 melanoma cells did not express theCD66a protein. Furthermore, no CD66a expression was observed among NKcells derived from the peripheral blood of 10 other melanoma patientswith no clinical evidence of active disease.

PBL from eight healthy donors were also obtained, and the expression ofCD66a on NK cells was analyzed using the same quadruple staining as thatdescribed above. Very little or no CD66a staining was observed among allNK cells tested (a representative healthy donor OM is shown in FIG.24↑D). This is in agreement with an other observation demonstrating theexpression of the CD66a molecule on activated NK clones only.

CD66a interactions are used by some melanoma cells as a mechanism ofdefense to avoid attack by CD66a-positive NK cells.

TABLE 6 Expression of various putative lysis ligands on human melanomacell lines Melanoma Cell Lines CD99-Ig CD16-Ig NKp30-Ig NKp44-IgNKp46-Ig L33melA1 0.0 0.20 2.71 3.28 0.52 L33melB1 0.0 1.14 8.76 6.411.45 1106mel 0.0 3.70 39.1 15.22 3.05 FO-1 0.0 2.44 12.32 13.47 2.261259mel 0.0 1.24 13.81 11.63 6.01 1074mel 0.0 1.16 12.42 24.35 2.441612mH 0.0 0.0 4.49 20.15 9.75 1612mel 0.0 0.1 2.53 15.28 2.71Melanoma cell lines were stained with various Ig fusion proteins asdescribed in Materials and Methods of this section. Data are presentedas MFI after subtraction of the background PE-conjugated anti-human Fcstaining and are representative of one experiment of three performed.

TABLE 7 Expression of class I MHC and CD66a proteins on human melanomacell lines Melanoma Cell Lines Background W632 Kat4c 1106mel 2.97 3.25200 1074mel 3.25 3.25 137.7 1259mel 1.60 1.60 108.3 1612mel 3.13 3.7935.5 FO-1 2.60 2.44 25.9 1612mH 3.79 3.82 25.4 L33melB1 2.89 78.4 15.5M-77 3.59 257 120 M-112 2.19 154 91 M-21 3.55 449 84.6 M-5 3.55 155567.5 M-139/1 4.68 1286 49.7 M-128 1.67 226 39 M-147 2.39 518 26.9 M-1455.1 1715 23.1 M-117 4.66 143 15.6 M-144 2.97 159 6.3 M-82 2.79 109 6.0M-139/2 5 1000 5.0 M-133 2.48 2308 3.02 L33melA1 2.81 132 3.16 M-90 1.891382 2.23Staining of class I MHC-negative and -positive melanoma cell lines wasperformed with the pan anti-class I mAb W632 and the anti-CD66 mAbKat4c. Similar staining levels of all melanoma cell lines were observedwhen the anti-CD66a mAb 5F4 was used. Data (MFI) are representative ofone experiment of three performed.

Example 5 Pivotal Role of CEACAM1 Protein in the Inhibition of ActivatedDecidual Lymphocyte Functions

Lymphocytes in direct contact with embryonic extravillous trophoblastsconstitute more than 40% of decidual cells and appear to play majorroles in implantation and early gestation. A unique subset of NK cells,making up 70-80% of decidual lymphocytes, express high levels of CD56but lack CD16. The present inventors have demonstrated a novel class IMHC-independent inhibitory mechanism of NK cell cytotoxicity that ismediated by CEACAM1 homotypic interactions. This mechanism is used bysome melanoma cells to avoid attack, mainly by CD16-NK cells. Thepresent invention demonstrate that CEACAM1 is expressed on primaryextravillous trophoblasts and is upregulated on the vast majority ofIL-2-activated decidual lymphocytes, including NK, T, and NKT cells. Inthis section it is shown that CEACAM1 interactions inhibit the lysis,proliferation, and cytokine secretion of activated decidual NK, T, andNKT cells, respectively. In vivo analysis of decidual lymphocytesisolated from cytomegalovirus-infected (CMV-infected) pregnant womenrevealed a dramatic increase in the expression of CEACAM1. It ispossible that a novel ligand for this adhesion molecule is present onthe surface of CMV-infected fibroblasts. This section demonstrates amajor role for the CEACAM1 protein in controlling local decidual immuneresponses. [Gal Markel et al., Pivotal role of CEACAM1 protein in theinhibition of activated decidual lymphocyte functions, The Journal ofClinical Investigation, 110:943-953 (2002). This reference is hereinincorporated by reference.]

During embryonic implantation, the extravillous trophoblast (EVT) cellsinvade the uterine endometrium. At this site, a direct-contact interfaceforms between maternal and embryonic cells, which locally modifies theproperties of the uterine mucosa. Embryonal-maternal interface togetherwith specialized ECM constitutes the decidua basalis. Remarkably, morethan 40% of decidual cells are immune cells. This suggests that thematernal immune system is involved in the modulation ofmaternal-embryonal interactions. The decidual lymphocyte compositiondiffers significantly from that of peripheral blood lymphocytes. Morethan 70% of decidual lymphocytes are CD56bright CD16− (FcRγIII) NKcells, while T cells constitute only 10%. In contrast, only 10% of theperipheral blood lymphocytes are NK cells that are characterized by amoderate expression level of the CD56 protein and the expression of theCD16 receptor. It is currently believed that decidual lymphocytes areimportant for control of normal trophoblastic growth, differentiation,and invasion. However, their role in combating pathogens in the contextof pregnancy is only poorly understood.

The gentle balance between immune tolerance and immune activation thatmight lead to the rejection of the embryo by the decidual lymphocytes ismaintained via several mechanisms, involving both decidual lymphocytesand EVTs. EVT invasion might be controlled by the modulation of thelocal cytokine profile, and therefore the cytokine release of deciduallymphocytes must be tightly regulated. The killing activity of both NKcells and CTLs, belonging to the innate and adaptive branches of theimmune system, respectively, is regulated by the class I MHC proteins.While the recognition of the class I MHC proteins by the T cellreceptors (TCRs) of CTLs activates T cell-mediated killing, theinteractions between NK cells and the same proteins suppress NK cellcytotoxicity. It was reported that EVTs express an unusual combinationof two nonclassical class I MHC proteins, the HLA-E and HLA-G, alongwith the classical HLA-C protein, but that they do not express the HLA-Aand HLA-B proteins. As most of the CTLs are directed against HLA-A and-B proteins, this unique pattern of expression of class I MHC proteinsprobably prevents rejection of the semiallogeneic fetus by CTLs. The HIVvirus uses a similar mechanism of specific downregulation of HLA-A and-B proteins, mediated by the Nef protein, to avoid attack by CTL.

NK cells compose the vast majority of decidual lymphocytes that are incontact with EVTs. The fetus is protected from rejection by maternal NKcells for several reasons. First, decidual NK cell inhibition appearsskewed toward HLA-C recognition, compared with peripheral blood NKcells. Fifty to eighty percent of decidual NK cells are inhibited byHLA-C, compared with only 5-20% of the peripheral blood NK cells.Second, virtually all decidual NK cells express the HLAE-bindinginhibitory receptor complex CD94/NKG2A five times more than doperipheral blood NK cells. Furthermore, the HLA-E protein, which isexpressed on cell surface upon binding of peptides derived from theleader sequence of various class I MHC proteins, binds, with thegreatest affinity, the leader peptides of HLA-G and HLA-C proteins,which are both expressed on the EVT cells. Third, all decidual NK cellsexpress the inhibitory LIR1 (ILT2) or KIR2DL4 receptors, both of whichare able to interact with the HLA-G proteins. Fourth, decidual NK cellshave decreased killing activity against class I MHC-negative targetcells. This wide spectrum of mechanisms aimed at controlling thecytolytic function of decidual NK cells further demonstrates theimportance of these cells in the rejection of allogeneic transplants. Italso implies that other mechanisms with the ability to control thefunction of decidual lymphocytes might exist.

The CEACAM1 protein, a member of the CEACAM family, is expressed on abroad spectrum of cells (13). It belongs to the Ig superfamily andinteracts in both a homotypic manner and a heterotypic manner with othervariants of the CEACAM family, including the CEACAM6 and CEACAM5proteins. The CEACAM1 homotypic interactions between NK cells andvarious target cells inhibit NK cytotoxicity. This novel class IMHC-independent mechanism appears to be used mainly by CD16-NK cells andmight play an important role in the development of various pathologies,such as melanoma.

It is shown that CEACAM1 is expressed by EVTs as well as by the majorityof IL-2-activated decidual lymphocyte subsets. The engagement of theCEACAM1 protein leads to the inhibition of NK killing, T cellproliferation, and IFN-γ secretion by NKT cells. The in vivoupregulation of the CEACAM1 protein on the majority of deciduallymphocytes might have an important role in controlling local immuneresponse. This is demonstrated by the analysis of decidual lymphocytesubsets obtained from decidua of cytomegalovirusinfected (CMV-infected)women, which revealed a dramatic upregulation of surface CEACAM1expression. In addition, evidence is provided that CEACAM1 binds andfunctionally interacts with an unidentified molecule present on humanprimary fibroblasts infected with the laboratory AD169 CMV strain orwith a clinical CMV strain isolated from infected decidua. Thesecombined results suggest a major role for the CEACAM1 protein incontrolling local decidual immune responses.

Methods Cells, Transfections, Virus Propagation, and Antiviral Agent

The cell lines used in this section were the class I MHC-negativeEpstein-Barr virus-transformed B cell line 721.221 (.221), .221 cellstransfected with the CEACAM1 cDNA, and the murine thymoma BW cell line,which lacks expression of α and β chains of the TCR. Stable transfectionof .221 cells expressing CEACAM6 and CEACAM5 was performed byelectroporation (0.23 kV, Cap uF] 250 uF). The cDNA for CEACAM6 wasamplified by RT-PCR and cloned into pcDNA3 expression vector, and theCEACAM5 cDNA was a kind gift from W. Zimmermann,Ludwig-Maximilians-University, Muenchen, Germany) Human foreskinfibroblasts (HFFs) were used for propagation and infection of human CMVstrain AD169 (American Type Culture Collection, Manassas, Va., USA), aspreviously described. After a 1-hour period of virus adsorption tocells, 300 ug/ml of the CMV DNA polymerase inhibitor phosphonoformate(PFA; Sigma-Aldrich, St. Louis, Mo., USA) was added for inhibition ofvirus replication.

Primary CMV Infection, Definition of Congenital CMV Infection, andTermination of Pregnancy

Primary CMV infection during pregnancy was diagnosed by documentation ofmaternal seroconversion, with appearance of CMV antibodies duringpregnancy in women known to be CMVseronegative before gestation.Diagnosis of CMV fetal infection was based on viral isolation (by shellviral culture and conventional culture) from amniotic fluid obtained atthe 22nd week of gestation, along with PCR detection of viral DNA in theamniotic fluid. Congenital disease could be predicted by the presence ofcharacteristic ultrasonographic findings including cerebralcalcifications and microcephaly. Decision to terminate pregnancy wasbased on documentation of fetal infection and disease. Deciduae fromfirst-trimester elective terminations were obtained by scraping.

Antibodies

The mAb's used in this section were FITCconjugated Kat4c mAb directedagainst CEACAM1, -5, and -6 (DAKO, Glostrup, Denmark),phycoerythrinconjugated anti-CD56 mAb (BD Pharmingen, San Diego, Calif.,USA), CyChrome-conjugated anti-CD3 mAb (BD Pharmingen), and biotinylatedanti-CD16 mAb (Serotec, Oxford, United Kingdom), followed byCy5-streptavidin (Jackson ImmunoResearch Laboratories Inc., West Grove,Pa., USA). The anti-CD4 mAb (DAKO), anti-Vβ3 (BD Pharmingen), anti-Vβ17(BD Pharmingen), and the anti-CEACAM1 5F4 mAb were also used. Forblocking assays, rabbit polyclonal anti-CEACAM1, -5, and -6 (DAKO)antibodies and the control rabbit polyclonal antibodies against purifiedubiquitin were used. The following anti-IFN-γ mAb's were purchased fromBD Pharmingen: mAb B27, used for measuring intracellular IFN-yproduction; and biotinylated mAb 4S.B3 (detection) and purified mAbHIB42 (capture), both used in the ELISA assays. The production of mouseIL-2 from BW/CEACAM1ξ-transfected cells was detected by ELISA usingpurified anti-mouse IL-2 mAb JES6-1A12 (capture) and biotinylatedanti-mouse IL-2 mAb JES6-5H4 (detection) (both from BD Pharmingen).ELISA assays were performed according the manufacturer's instructions(BD Pharmingen).

Isolation of Decidual Lymphocytes

The Hadassah Medical Organization Institutional Board approved obtainingdeciduae from elective pregnancy-termination procedures, from inducedlabors, and from caesarian sections, in keeping with the principles ofthe Helsinki Declaration. The tissue was trimmed into 1-mm pieces andenzymatically digested for 20 minutes, using vigorous shaking, with 1.5mg type I DNase and 24 mg type IV collagenase present in 15 ml ofRPMI-1640 medium. This procedure was repeated three times. After anadditional 5 minutes' incubation at room temperature without shaking,the supernatants were collected and incubated overnight in a tissueculture dish. Nonadherent cells were collected and loaded on Ficolldensity gradient to purify the lymphocyte population. Cells were furtheranalyzed by flow cytometry. NK and NKT cells were purified usinganti-CD56 mAb followed by incubation with microbeads of conjugated goatanti-mouse IgG antibodies (Miltenyi Biotec Inc., Auburn, Calif., USA).Separation was performed with the AutoMACS instrument (Miltenyi BiotecInc.). Positive (NK and NKT cells) and negative (T cells) fractions werecollected and cloned (one cell per well) in the presence of IL-2.

Quadruple Staining

For quadruple staining, the following fluorochrome-conjugated mAb's wereused: FITCconjugated anti-CEACAM Kat4c mAb (DAKO),phycoerythrin-conjugated anti-CD56 mAb (BD Pharmingen), andCyChrome-conjugated anti-CD3 mAb (BD Pharmingen). As the fourth color,biotinylated anti-CD16 mAb (Serotec) was used, followed byCy5-streptavidin (Jackson ImmunoResearch Laboratories Inc.) as a secondreagent. To block nonspecific binding, cells were first incubated for 1hour on ice with 25% human serum, and then incubated with the variousantibodies.

Cytotoxicity Assays

The cytotoxic activity of NK cells against the various targets wasassayed in 5-hour 35Srelease assays, as described previously. Briefly,cells were labeled overnight with 35S-methionine and washed, and 5·103labeled target cells were incubated at various effector-to-targetratios. The killing rate was calculated as percent 35S-methioninerelease=(cpm sample−cpm−spontaneous release)/(cpm total−cpm spontaneousrelease)×100. Total 35S-methionine release was measured after incubationof the cells with 0.1 M NaOH. In all presented cytotoxic assays, thespontaneous release was less than 25% of maximal release. In experimentswhere mAb's were included, the final mAb concentration was 10 ug/ml, or40 ul/ml in those cases where rabbit polyclonal antibodies were used.

Staphylococcal Enterotoxin B-Induced T Cell Proliferation

These assays were performed as previously described. Briefly, target.221 and .221/CEACAM1 cells were irradiated (60 Gy). Thereafter, 50,000T cells, 25,000 target cells, and various concentrations of superantigenwere mixed in a total volume of 200 ul of RPMI-10% FCS in each well of a96-well plate. After incubation at 37° C. and 5% CO2 for 2 days, 1 uCiof 3H-thymidine was added to each well and the cells were furtherincubated at 37° C. overnight. The cells were then harvested and countedon a liquid scintillation counter (1450 Micro-Beta PLUS; Wallac, Turku,Finland). In analysis of the cpm from each well, the background cpm fromwells in which identical reagents and target cells were placed in theabsence of any T cells was subtracted.

Generation of IG Fusion Proteins

The extracellular portion of the CEACAM1 protein was amplified by PCRusing the following primers: 5′-CCCAAGCTTGGGGCCGCCACCATGGGGCACCTCTCAGCC(including HindIII restriction site) and 3′-GCGGATCCCCAGGTGAGAGGC(including BamHI restriction site). A silent mutation, adenine 885guanidine (no change in glycine 281), was performed by site-directedmutagenesis to cancel the BamHI site in the amplified sequence. Thegeneration, production, and staining procedures of the Ig fusionproteins were previously described. [Gal Markel et al., Pivotal role ofCEACAM1 protein in the inhibition of activated decidual lymphocytefunctions, The Journal of Clinical Investigation, 110:943-953 (2002).This reference is herein incorporated by reference.] Briefly, thePCR-generated fragments were cloned into a mammalian expression vectorcontaining the Fc portion of human IgG1 (a kind gift from B. Seed,Massachusetts General Hospital, Department of Molecular Biology, Boston,Mass., USA). Sequencing of the constructs revealed that all cDNAs werein frame with the human Fc genomic DNA and were identical to thereported sequences. COS-7 cells were transiently transfected with theplasmids containing cDNAs using FuGENE6 reagent (Roche MolecularBiochemicals, Indianapolis, Ind., USA) according to the manufacturer'sinstructions, and supernatants were collected and purified on a proteinG column. SDS-PAGE analysis revealed that all Ig fusion proteins wereapproximately 95% pure and of the proper molecular mass. To assay forthe CEACAM binding, various cells were incubated with 50 ug/ml of fusionprotein for 2 hours on ice. The cells were washed and incubated with Fcfragment-specific (minimal cross-reaction to bovine, horse, and mouseserum proteins), phycoerythrin-conjugated affinity-purified F(ab2)2fragment of goat anti-human IgG (Jackson ImmunoResearch LaboratoriesInc.). Incubation was performed for 1 hour and analyzed by flowcytometry with a FACScan (Becton Dickinson Immunocytometry Systems, SanJose, Calif., USA).

Generation of BW Cells Expressing the Chimeric Ceacam1 Protein and theProduction of IL-2

The extracellular portion of the human CEACAM1 protein was amplified byPCR using the following primers:5′-CCCAAGCTTGGGGCCGCCACCATGGGGCACCTCTCAGCC (including HindIIIrestriction site) and 3′-GTAGCAGAGAGGTGAGAGGCCATTTTCTTG (including firstnine nucleotides of mouse ξ chain transmembrane portion). The mouse ξchain was amplified by PCR using the following primers:5′-CTCTCACCTCTCTGCTACTTGCTAGATGGA (including last nine nucleotides ofhuman CEACAM1 extracellular portion) and3′-GGAATTCCTTAGCGAGGGGCCAGGGTCTG (including EcoRI restriction site). Thetwo amplified fragments were mixed, and PCR was performed with the52€HindIII primer and the 32 EcoRI primer for the generation of theCEACAM1

construct. The CEACAM1

€construct was cloned into pcDNA3 expression vector (Invitrogen Corp.,Carlsbad, Calif., USA) and stably transfected into BW cells. Formeasurement of IL-2 production resulting from the homotypic CEACAM1interactions, 50,000 BW or BW-transfected cells were incubated inRPMI-10% FCS medium for 48 hours at 37° C. and 5% CO2. Supernatants werecollected and the presence of IL-2 was monitored by using anti-IL-2 mAband standard ELISA assays (BD Pharmingen). For measurement of IL-2production resulting from the CEACAM1 interactions of different celltypes, 50,000 BW or BW-transfected cells were incubated in RPMI-10% FCSwith irradiated .221 or with .221/CEACAM1 cells for 24 hours or withCMVinfected HFF cells for 48 hours at 37° C. and 5% CO2. The presence ofmouse IL-2 in cell supernatants was measured as above.

Cross-Linking of NKT Cells

NKT cells (105 per well) were incubated with or without 0.5

g of Kat4c mAb on ice for 1.5 hours in 96 round bottom microplates(Nalge Nunc, Rochester, N.Y., USA). Treated NKT cells, present in 200 ulof IL-2-containing medium, were then cultured in 96 flat bottommicroplates (Nalge Nunc) precoated with 1 ug/well of sheep antimouse IgGantibodies (ICN Biomedicals Inc., Costa Mesa, Calif., USA) for 24 hoursat 37° C. Cells were then analyzed by FACS.

Permeabilization and Intracellular IFN-γ Staining

The permeabilization and intracellular IFN-© staining were performedusing the Cytofix/Cytoperm Plus (with GolgiStop) kit (BD Pharmingen)according to the manufacturer's instruction.

Results CEACAM1 is Expressed on Different Decidual Lymphocytes afterActivation

To test the possible role of CEACAM1 in controlling decidual lymphocytefunctions, decidual lymphocytes from first-trimester elective pregnancyterminations were isolated as described in Methods. Obtained tissueswere identified as decidua by histologic analysis. Lymphocytes wereisolated from nine different deciduae and quadruple-stained using flowcytometry for the expression of CD3, CD16, CD56, and CEACAM. Inagreement with previous observations, the total decidual lymphocytepopulation contained mainly CD16− NK cells (70-80%, characterized byCD3− CD56bright), but T (characterized by CD3+CD56−) and NKT(characterized by CD3+CD56+) cells were also identified (5.3% and 3.2%,respectively). Little or no staining for the CEACAM1 protein wasobserved among all decidual lymphocyte populations tested (FIG. 25,a-c).

Various lymphocytes were cloned and cultured for 3 weeks in the presenceof IL-2 (50 U/ml). Remarkably, staining with the 5F4 anti-CEACAM1 mAb(see FIG. 26) revealed a dramatic increase in the CEACAM1 proteinexpression on the surface of the vast majority of NK, T, and NKT cellclones tested (85%, 86%, and 95%, respectively; surface expression ofCEACAM1 on representative clones is shown in FIG. 25, d-f). This is inmarked contrast to NK cells derived from peripheral blood, in whichsurface CEACAM1 expression could be detected on only 2-3% ofIL-2-activated CD16+NK clones and on about 45% of the IL-2-activatedCD16− clones. Notably, the expression levels of the CEACAM1 on thesurface of all tested clones were more than threefold above background.This level of expression was reported to be sufficient for effectiveinhibition of NK cytotoxicity.

As the CEACAM1 protein interacts homotypically with other CEACAM1proteins (see FIGS. 30 and 31) and decidual lymphocytes are in directcontact with embryonic EVT cells in vivo, it was important to testwhether EVT cells express the CEACAM1 protein. EVT cells were obtainedfrom the same elective pregnancy terminations from which deciduallymphocytes were isolated and were tested for the expression of HLA-Gand CEACAM1. As the expression of HLA-G is restricted to EVT cells only,isolated cells were identified as EVT cells by using specific stainingwith the anti-HLA-G specific mAb MEM-G/13B. The mAb MEMG/13Bspecifically stains the class I MHC-negative .221 cells transfected withHLA-G; it did not stain .221 cells transfected with other class I MHCcDNA. FACS staining analysis of isolated EVT cells showed that thesecells express the HLA-G (FIG. 25g ) and the CEACAM1 (FIG. 25h )proteins. These findings suggest that CEACAM1 might mediate directinteractions between activated decidual lymphocytes and EVTs and thusmight display a novel control mechanism protecting the embryo fromsustaining damage.

CEACAM1 Interactions Inhibit Decidual NK Cytotoxicity

It has been demonstrated that the CEACAM1-mediated inhibition of NKcells can be blocked by using rabbit polyclonal anti-CEACAM antibodiesand not by the mAb 5F4 or the mAb Kat4c [Markel, G., et al. 2002. CD66ainteractions between human melanoma and NK cells: a novel class IMHC-independent inhibitory mechanism of cytotoxicity. J. Immunol.168:2803-2810.158:11-25. This reference is incorporated by reference.].It is shown that the CEACAM1 protein interacts with other CEACAMproteins, such as CEACAM5 and CEACAM6, and that the binding site ofCEACAM1 was located at the N-terminal Ig-V-type domain of the CEACAM1protein (23). The N-terminal Ig-V-type domain of the CEACAM familyreveals 70-90% sequence similarity among the different variants. It wastherefore important to determine the specificity of all anti-CEACAM1antibodies used in this work. .221 cells were transfected with CEACAM1,CEACAM6, and CEACAM5 and stained for surface expression using thevarious anti-CEACAM antibodies.

FIG. 26 shows that all anti-CEACAM antibodies specifically recognizedmembers of the CEACAM family. This is because no staining was observedon either nontransfected .221 cells or the control HLAB27-transfected.221 cells. The 5F4 mAb recognized the CEACAM1 protein only, whereas theKat4c mAb and the rabbit polyclonal antibodies directed against CEACAMrecognized CEACAM1, CEACAM6, and CEACAM5 proteins (FIG. 26).

To investigate whether the CEACAM1 protein is functional, IL-2-activateddecidual NK clones, expressing the CEACAM1 protein (a representativeclone is shown in FIG. 27a ), were tested in killing assays against .221cells and .221 cells transfected with CEACAM1 (.221/CEACAM1). Thegeneration of these transfectants was described previously (15). TheCEACAM1+NK clones effectively killed .221 cells, whereas inhibition oflysis was observed when .221/CEACAM1 cells were used (FIG. 27b ). Theinhibition of NK killing by .221/CEACAM1 cells was the result of theCEACAM1 homotypic interactions, as lysis of .221/CEACAM1 cells wasrestored when rabbit anti-human CEACAM antibodies were included in theassay. The addition of a control rabbit serum derived fromubiquitin-immunized rabbit had no effect. No difference in the lysis of.221 or .221/CEACAM1 cells was observed when CEACAM1− NK clones wereused. Most decidual NK clones displayed only limited cytotoxicityagainst the .221 target cells (10-20% lysis). The killing of.221/CEACAM1 cells by “low killer” decidual NK clones was also decreasedbecause of the homotypic CEACAM1 interactions, and the addition ofanti-CEACAM polyclonal antibodies restored lysis.

CEACAM1 Interactions Inhibit Staphylococcal Enterotoxin B-InducedDecidual T Cell Proliferation

As the expression of CEACAM1 protein was also demonstrated on the vastmajority of T lymphocytes activated by IL-2, the effect of CEACAM1interactions on T cell proliferation was also tested. Superantigens caninduce T cell proliferation by binding to class II MHC proteins andspecific TCR VP chains. The staphylococcal enterotoxin B (SEB)superantigen interacts with various TCR VP chains, including Vβ3 andVβ17. Decidual T cell clones were obtained as described in Methods andscreened by flow cytometry for the expression of CD4, Vβ3, and Vβ17 byusing specific mAb's. A representative T cell clone, no. 1, stainedbrightly for both CD4 and Vβ17 (FIG. 28, a and b), and moderately forCEACAM1 (FIG. 28c ). The SEB-induced proliferation of this T cell clonewas assayed as described in Methods. A dramatic increase in the T cellproliferation was observed when cells were incubated with .221 cells inthe presence of 250 ng/ml of SEB (50-fold above the backgroundproliferation without SEB; data not shown). Efficient inhibition of theT cell proliferation (around 50%) was observed in all SEB concentrationstested when cloned T cells were incubated with .221/CEACAM1 cells (FIG.28d ). The expression levels of the class II MHC proteins were similaron both .221 and .221/CEACAM1 cells.

CEACAM1 Interactions Inhibit Secretion of Cytokines From Decidual NKTCells

Cytokines might play an important role in fetus development. NKT cellsthat are present among the decidual lymphocyte population (see FIG. 25)are able to produce large amount of cytokines. The functional effect ofCEACAM1 interactions on cytokine secretion of decidual NKT cells hasnever been investigated. Decidual NKT clones were cultured as describedin Methods and screened for CEACAM1 expression by flow cytometry, usingthe anti-CEACAM1 5F4 mAb (a representative NKT cell clone, no. 3, isshown in FIG. 29a ). NKT clone 3 spontaneously secreted IFN-γ into themedia, as measured by ELISA (FIG. 29b ). Other cytokines such as IL-4,IL-5, IL-13, TNF, and macrophage inflammatory protein-1α could not bedetected in culture supernatant of this clone. Cross-linking of CEACAM1for 24 hours with the Kat4c mAb dramatically decreased the amount ofIFN-γ detected in the medium of this NKT cell clone (FIG. 29b ). Inorder to determine whether the inhibitory effect observed aftercross-linking of CEACAM1 on NKT cells is the result of decreasedsecretion or decreased production of IFN-γ, the presence ofintracellular IFN-γ, before and after cross-linking of CEACAM1, wastested by staining as described in Methods. Untreated NKT cells showedlittle staining for intracellular IFN-γ (median fluorescence intensitytwofold above background; FIG. 29c ). After cross-linking with the Kat4cmAb, the staining for intracellular IFN-γ increased significantly(median fluorescence intensity 4.5-fold above background; FIG. 29d ).These findings suggest that CEACAM1 engagement on NKT cells suppressesthe cytokine secretion machinery and not de novo synthesis.

In Vivo Upregulation of CEACAM1 on Decidual Lymphocytes

The above observations suggest a major role for the CEACAM1 protein inthe regulation of decidual lymphocyte functions after IL-2 activation.In vivo activation of decidual lymphocytes might occur as a result ofviral infection. CMV is the leading cause of congenital viral infectionsin Western countries. It was therefore tested whether CEACAM1 expressioncould be detected on the surface of lymphocytes obtained from deciduaeof women who had primary CMV infection during gestation with documentedintrauterine manifestations. Second and third-trimester pregnancyterminations of women diagnosed with primary CMV infection necessitatethe administration of labor-promoting agents that might have someimmunological effects. To control the experiment, the expression ofCEACAM1 protein on the surface of lymphocytes obtained either fromthird-trimester caesarian sections with labor (Table 1; this section) orfrom the deciduae taken from caesarian sections without labor (Table 1;this section) were analyzed. Decidual lymphocytes were obtained andstained for the presence of CEACAM1 on NK, NKT, and T cells as above.Only very limited numbers of NKT cells were isolated, and therefore theexpression of CEACAM1 on NKT cells could not be determined. Remarkably,a significant elevation of CEACAM1 expression was observed in NK and Tcells obtained from deciduae of CMV-infected women, whereas little or noexpression of CEACAM1 was observed in the two control groups (Table 1;this section). CEACAM1 expression can vary significantly betweendifferent CMV-infected deciduae. In one patient, 90% and 95% of the NKand T cells, respectively, expressed the CEACAM1 protein, whereas in thesecond patient the expression of the CEACAM1 protein was limited to 10%and 10.2% of NK and T cells, respectively. However, the expression ofthe CEACAM1 protein, even in the second patient, was still verysignificant compared with that of the control groups, and it was similarto the expression level of other class I MHC inhibitory receptors, whichvary between 5% and 20%. There are several possible reasons for thedifferences in the level of CEACAM1 expression, such as subjective localimmune response, course of CMV infection, and the time of pregnancytermination after the initiation of infection. The mild expression ofthe CEACAM1 protein on trophoblasts obtained from normal decidua (FIG.25h ) was still maintained on trophoblasts obtained from infecteddecidua.

CMV-Infected Fibroblasts Express a Novel Ligand for CEACAM1

The results presented above demonstrate that CEACAM1 expression isupregulated in vivo in lymphocytes obtained from CMV-infected deciduae.Expression of CEACAM1 was observed on EVT cells obtained from eithernormal or CMV-infected deciduae (FIG. 25). CEACAM1 homotypicinteractions might occur in vivo, leading to lymphocyte inhibition.

Only two cases of CMV-infected deciduae are presented here, as studiesin vivo are limited for several reasons. In addition to the fact thatprimary CMV infection during pregnancy is quite rare, the detection anddiagnosis are quite difficult. Furthermore, deciduae from CMV-infectedwomen were used only if they spontaneously detached, to avoidunnecessary additional procedures. However, in both presented cases,CEACAM1 upregulation was observed. To further establish the effect ofCMV infection with regard to CEACAM1 inhibition and to test whether theCMV uses the CEACAM1 inhibitory mechanism to avoid attack by the immunesystem, CMV-infected HFFs were used. HFF cells were infected with CMVstrain AD169 with moi 2-3. No staining of either infected or uninfectedHFF cells with anti-CEACAM1, -5, and -6 Kat4c mAb was observed at anytime point before or after the infection. Infected cells were harvestedat different time points at 24-hour intervals after the infection andstained for the presence of CEACAM1 ligand using CEACAM1-Ig fusionprotein, as described in Methods of this section.

The CEACAM1-Ig fusion protein specifically stained the .221/CEACAM1cells and did not stain the .221 cells (FIG. 30a ), indicating thatCEACAM1 homotypic interactions are strong enough to be detected by thismethod. No staining of CEACAM1-Ig was observed in the first 4 days afterthe infection (FIG. 30b ). CEACAM1-Ig staining was observed starting onday 5 and reaching maximum on day 6 after the infection. All infectedcells were positively stained with anti-pp65 mAb. The CEACAM1-Ig bindingobserved was only to the HFF-infected cells, not to the uninfectedcells. No changes in the level of the control CD99-Ig fusion proteinstaining were observed at any time point (FIG. 30b ). As CEACAM1 caninteract only with the CEACAM1, -5, and -6 variants, and as it was alsoreported that CEACAM variants cannot be detected on the surface of humanfibroblasts, suggesting the existence of a novel ligand for CEACAM1 onthe surface of CMV-infected HFF cells. This novel ligand appears lateafter the infection. To further test this hypothesis, similarexperiments in the presence of the antiviral agent PFA, which is knownto block viral DNA synthesis and earlylate-phase transition wereperformed. Progeny virus titers in culture supernatants were determinedon day 4 after infection by a standard plaque titration assay on HFFs.In the absence of PFA, virus titer was 3×106 plaque-forming units/ml,whereas in the presence of PFA no virus could be detected. In agreementwith the above observations demonstrating the appearance of CEACAM1ligand on the surface of CMV-infected HFFs, the addition of PFAcompletely abolished the binding of CEACAM1-Ig to the infected HFF cells(FIG. 30b ).

Whether the CEACAM1 interactions with the CMV-infected HFFs arefunctional was tested. Mouse BW cells were stably transfected with achimeric molecule composed of the extracellular portion of CEACAM1 fusedto mouse

chain (as described in Methods). Engagement of CEACAM1 leads to thesecretion of mouse IL-2, mediated by the

chain. The IL-2 amounts in the cell supernatants can be measured byELISA. Secretion of IL-2 could be detected in the culture supernatantsof the BW cells transfected with CEACAM1

, but not in the culture supernatants of the BW cells or BW cellstransfected with CD16

(FIG. 31a ). Moreover, IL-2 secretion was also detected in thesupernatants of BW/CEACAM1

cells when cells were incubated with .221/CEACAM1 cells, but not withthey were incubated with .221 cells (FIG. 31b ). Thus, homotypic CEACAM1interactions are strong enough to induce IL-2 secretion in this system.In agreement with the CEACAM1-Ig staining data, efficient secretion ofIL-2 was observed (on days 5 and 6 after the infection) in thesupernatants of BW/CEACAM1

cells cultured with infected HFFs. This IL-2 secretion was blocked bythe addition of PFA (FIG. 31c ). No IL-2 secretion was observed in theculture supernatants of BW/CD16

cells incubated with uninfected or infected HFF cells.

To further substantiate the above results, the clinical CMV strainisolated from the infected decidua was cultured (patient 6; Table 1 ofthis section) with infected HFF cells. The propagation of the virus wasmuch slower than that of the laboratory AD169 strain. Consistentmicroscopic monitoring of infected HFF cells revealed that even afterprolonged propagation time, only partial infection could be achieved.One month after initiation of infection, infected HFF cells wereanalyzed for recognition by CEACAM1. HFF cells were stained withIg-fused proteins, including CEACAM1-Ig and the control CD99-Ig. Nostaining of uninfected HFF cells was observed. Specific staining of theinfected HFF cells could be observed with the CEACAM1-Ig but not withthe CD99-Ig (20% and 2% staining, respectively; FIG. 32a ). No stainingwas observed when anti-CEACAM antibodies were used, suggesting thatCEACAM1-Ig recognizes a novel CMV-induced ligand on infected HFFs.Whether this recognition is capable of eliciting IL-2 secretion fromBW/CEACAM1

cells was tested. IL-2 levels were measured in the supernatants of BW orBW/CEACAM1

cells cocultured with HFF cells infected with the clinical CMV strainisolated from patient 6. In agreement with the CEACAM1-Ig staining,increased IL-2 secretion could be detected only in the supernatants ofthe BW/CEACAM1

cells coincubated with infected HFF cells (FIG. 32b ). The moderateelevation of IL-2 secretion and the partial staining of CEACAM1-Ig (FIG.32, a and b) are correlated with the low infection levels of thisclinical CMV strain observed in vitro, and with the moderate percentagesof CEACAM1+ lymphocytes isolated from infected decidua no. 2 (patient 6;Table 1 of this section). Similar results were obtained with anotherclinical CMV strain, isolated from a neonate's urine.

TABLE 1 CEACAM1 expression is upregulated on decidual lymphocytes fromwomen with primary CMV infection Patient no. Decidua source NK cells Tcells NKT cells 1 With labor 0.5%  1.9% Not detected 2 With labor  1% 0% Not detected 3 Without labor 1.7%  2.5% Not detected 4 Without labor 0% 0.4% Not detected 5 With CMV 95%  90% Not detected 6 With CMV 10%10.2%  Not detectedCells were isolated from deciduae from different groups andquadruplestained for CD3, CD16, CD56, and CEACAM1 as described inMethods. The percentage of CEACAM1+ cells of each indicated lymphocytesubset is shown.

What is claimed is:
 1. A method for inducing a protective immunity in atarget tissue, wherein said method comprises the induction of CEACAM1protein production in said target tissue.
 2. The method of claim 1,wherein said target tissue comprises tissue afflicted by an autoimmunedisease.
 3. The method of claim 1, wherein said target tissue comprisestissue being prepared for transplantation.
 4. The method of claim 1,wherein said induction of CEACAM1 protein production comprises theactivation of CEACAM1 gene expression, said activation comprisingcontacting said target tissue with a signal transduction protein,transcriptional activator protein, nucleic acid, small moleculecompound, or combination thereof.
 5. The method of claim 1, wherein saidinduction of CEACAM1 protein production comprises the transfer of anucleic acid sequence into the cells of said target tissue, said nucleicacid encoding the CEACAM1 protein.
 6. The method of claim 1, whereinsaid induction of CEACAM1 protein production comprises the transfer of anucleic acid sequence into the cells of said target tissue, said nucleicacid encoding a protein that induces CEACAM1 mRNA expression.
 7. Themethods of claims 5 and 6, wherein said transfer of nucleic acid intothe cells of said target tissue comprises viral-mediated transfer,particle-mediated transfer, or magnetic cationic liposome mediatedtransfer.
 8. A method for inducing a protective immunity in a targettissue, wherein said method comprises the induction of CEACAM1 proteinproduction in said target tissue.